Artificial antibody polypeptides

ABSTRACT

The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

[0001] Portions of the present invention were made with support of theUnited States Government via a grant from the National Institutes ofHealth under grant number GM 55042 The U.S. Government therefore mayhave certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of theproduction and selection of binding and catalytic polypeptides by themethods of molecular biology. The invention specifically relates to thegeneration of both nucleic acid and polypeptide libraries encoding themolecular scaffolding of a modified Fibronectin Type III (Fn3) molecule.The invention also relates to “artificial mini-antibodies” or“monobodies,” i.e., polypeptides containing an Fn3 scaffold onto whichloop regions capable of binding to a variety of different molecularstructures (such as antibody binding sites) have been grafted.

BACKGROUND OF THE INVENTION

[0003] Antibody structure

[0004] A standard antibody (Ab) is a tetrameric structure consisting oftwo identical immunoglobulin (Ig) heavy chains and two identical lightchains. The heavy and light chains of an Ab consist of differentdomains. Each light chain has one variable domain (VL) and one constantdomain (CL), while each heavy chain has one variable domain (VH) andthree or four constant domains (CH) (Alzari et al., 1988). Each domain,consisting of˜110 amino acid residues, is folded into a characteristicβ-sandwich structure formed from two β-sheets packed against each other,the immunoglobulin fold. The VH and VL domains each have threecomplementarity determining regions (CDR1-3) that are loops, or turns,connecting β-strands at one end of the domains (FIG. 1: A, C). Thevariable regions of both the light and heavy chains generally contributeto antigen specificity, although the contribution of the individualchains to specificity is not always equal. Antibody molecules haveevolved to bind to a large number of molecules by using six randomizedloops (CDRs). However, the size of the antibodies and the complexity ofsix loops represents a major design hurdle if the end result is to be arelatively small peptide ligand.

[0005] Antibody substructures

[0006] Functional substructures of Abs can be prepared by proteolysisand by recombinant methods. They include the Fab fragment, whichcontains the VH-CH1 domains of the heavy chain and the VL-CL1 domains ofthe light chain joined by a single interchain disulfide bond, and the Fvfragment, which contains only the VH and VL domains. In some cases, asingle VH domain retains significant affinity (Ward et al., 1989). Ithas also been shown that a certain monomeric κ light chain willspecifically bind to its cognate antigen. (L. Masat et al., 1994).Separated light or heavy chains have sometimes been found to retain someantigen-binding activity (Ward et al., 1989). These antibody fragmentsare not suitable for structural analysis using NMR spectroscopy due totheir size, low solubility or low conformational stability.

[0007] Another functional substructure is a single chain Fv (scFv), madeof the variable regions of the immunoglobulin heavy and light chain,covalently connected by a peptide linker (S-z Hu et al., 1996). Thesesmall (M_(r) 25,000) proteins generally retain specificity and affinityfor antigen in a single polypeptide and can provide a convenientbuilding block for larger, antigen-specific molecules. Several groupshave reported biodistribution studies in xenografted athymic mice usingscFv reactive against a variety of tumor antigens, in which specifictumor localization has been observed. However, the short persistence ofscFvs in the circulation limits the exposure of tumor cells to thescFvs, placing limits on the level of uptake. As a result, tumor uptakeby scFvs in animal studies has generally been only 1-5% ID/g as opposedto intact antibodies that can localize in tumors ad 30-40% ID/g and havereached levels as high as 60-70% ID/g.

[0008] A small protein scaffold called a “minibody” was designed using apart of the Ig VH domain as the template (Pessi et al., 1993).Minibodies with high affinity (dissociation constant (K_(d))˜10⁻⁷ M) tointerleukin-6 were identified by randomizing loops corresponding to CDR1and CDR2 of VH and then selecting mutants using the phage display method(Martin et al., 1994). These experiments demonstrated that the essenceof the Ab function could be transferred to a smaller system. However,the minibody had inherited the limited solubility of the VH domain(Bianchi et al., 1994).

[0009] It has been reported that camels (Camelus dromedarius) often lackvariable light chain domains when IgG-like material from their serum isanalyzed, suggesting that sufficient antibody specificity and affinitycan be derived form VH domains (three CDR loops) alone. Davies andRiechmann recently demonstrated that “camelized” VH domains with highaffinity (K_(d)˜10⁻⁷ M) and high specificity can be generated byrandomizing only the CDR3. To improve the solubility and suppressnonspecific binding, three mutations were introduced to the frameworkregion (Davies & Riechmann, 1995). It has not been definitively shown,however, that camelization can be used, in general, to improve thesolubility and stability of VHs.

[0010] An alternative to the “minibody” is the “diabody.” Diabodies aresmall bivalent and bispecific antibody fragments, i.e., they have twoantigen-binding sites. The fragments contain a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) on thesame polypeptide chain (V_(H)-V_(L)). Diabodies are similar in size toan Fab fragment. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or “diabodies,”are bivalent and bispecific (P. Holliger et al., 1993).

[0011] Since the development of the monoclonal antibody technology, alarge number of 3D structures of Ab fragments in the complexed and/orfree states have been solved by X-ray crystallography (Webster et al.,1994; Wilson & Stanfield, 1994). Analysis of Ab structures has revealedthat five out of the six CDRs have limited numbers of peptide backboneconformations, thereby permitting one to predict the backboneconformation of CDRs using the so-called canonical structures (Lesk &Tramontano, 1992; Rees et al., 1994). The analysis also has revealedthat the CDR3 of the VH domain (VH-CDR3) usually has the largest contactsurface and that its conformation is too diverse for canonicalstructures to be defined; VH-CDR3 is also known to have a largevariation in length (Wu et al., 1993). Therefore, the structures ofcrucial regions of the Ab-antigen interface still need to beexperimentally determined.

[0012] Comparison of crystal structures between the free and complexedstates has revealed several types of conformational rearrangements. Theyinclude side-chain rearrangements, segmental movements, largerearrangements of VH-CDR3 and changes in the relative position of the VHand VL domains (Wilson & Stanfield, 1993). In the free state, CDRs, inparticular those which undergo large conformational changes uponbinding, are expected to be flexible. Since X-ray crystallography is notsuited for characterizing flexible parts of molecules, structuralstudies in the solution state have not been possible to provide dynamicpictures of the conformation of antigen-binding sites.

[0013] Mimicking the antibody-binding site

[0014] CDR peptides and organic CDR mimetics have been made (Dougall etal., 1994). CDR peptides are short, typically cyclic, peptides whichcorrespond to the amino acid sequences of CDR loops of antibodies. CDRloops are responsible for antibody-antigen interactions. Organic CDRmimetics are peptides corresponding to CDR loops which are attached to ascaffold, e.g., a small organic compound.

[0015] CDR peptides and organic CDR mimetics have been shown to retainsome binding affinity (Smyth & von Itzstein, 1994). However, asexpected, they are too small and too flexible to maintain full affinityand specificity. Mouse CDRs have been grafted onto the human Igframework without the loss of affinity (Jones et al., 1986; Riechmann etal., 1988), though this “humanization” does not solve theabove-mentioned problems specific to solution studies.

[0016] Mimicking natural selection processes of Abs

[0017] In the immune system, specific Abs are selected and amplifiedfrom a large library (affinity maturation). The processes can bereproduced in vitro using combinatorial library technologies. Thesuccessful display of Ab fragments on the surface of bacteriophage hasmade it possible to generate and screen a vast number of CDR mutations(McCafferty et al., 1990; Barbas et al., 1991; Winter et al., 1994). Anincreasing number of Fabs and Fvs (and their derivatives) is produced bythis technique, providing a rich source for structural studies. Thecombinatorial technique can be combined with Ab mimics.

[0018] A number of protein domains that could potentially serve asprotein scaffolds have been expressed as fusions with phage capsidproteins. Review in Clackson & Wells, Trends Biotechnol. 12:173-184(1994). Indeed, several of these protein domains have already been usedas scaffolds for displaying random peptide sequences, including bovinepancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)),human growth hormone (Lowman et al., Biochemistry 30:10832-10838(1991)), Venturini et al., Protein Peptide Letters 1:70-75 (1994)), andthe IgG binding domain of Streptococcus (O'Neil et al., Techniques inProtein Chemistry V (Crabb, L,. ed.) pp. 517-524, Academic Press, SanDiego (1994)). These scaffolds have displayed a single randomized loopor region.

[0019] Researchers have used the small 74 amino acid α-amylase inhibitorTendamistat as a presentation scaffold on the filamentous phage M13(McConnell and Hoess, 1995). Tendamistat is a β-sheet protein fromStreptomyces tendae. It has a number of features that make it anattractive scaffold for peptides, including its small size, stability,and the availability of high resolution NMR and X-ray structural data.Tendamistat's overall topology is similar to that of an immunoglobulindomain, with two β-sheets connected by a series of loops. In contrast toimmunoglobulin domains, the β-sheets of Tendamistat are held togetherwith two rather than one disulfide bond, accounting for the considerablestability of the protein. By analogy with the CDR loops found inimmunoglobulins, the loops the Tendamistat may serve a similar functionand can be easily randomized by in vitro mutagenesis.

[0020] Tendamistat, however, is derived from Streptomyces tendae. Thus,while Tendamistat may be antigenic in humans, its small size may reduceor inhibit its antigenicity. Also, Tendamistat's stability is uncertain.Further, the stability that is reported for Tendamistat is attributed tothe presence of two disulfide bonds. Disulfide bonds, however, are asignificant disadvantage to such molecules in that they can be brokenunder reducing conditions and must be properly formed in order to have auseful protein structure. Further, the size of the loops in Tendamistatare relatively small, thus limiting the size of the inserts that can beaccommodated in the scaffold. Moreover, it is well known that formingcorrect disulfide bonds in newly synthesized peptides is notstraightforward. When a protein is expressed in the cytoplasmic space ofE. coli, the most common host bacterium for protein overexpression,disulfide bonds are usually not formed, potentially making it difficultto prepare large quantities of engineered molecules.

[0021] Thus, there is an on-going need for small, single-chainartificial antibodies for a variety of therapeutic, diagnostic andcatalytic applications. In particular, there is an on-going need forartificial antibodies that are structurally stable at neutral pH.

SUMMARY OF THE INVENTION

[0022] The present invention provides a fibronectin type III (Fn3)molecule, wherein the Fn3 contains a stabilizing mutation. A stabilizingmutation is defined herein as a modification or change in the amino acidsequence of the Fn3 molecule, such as a substitution of one amino acidfor another, that increases the melting point of the molecule by morethan 0.1° C. as compared to a molecule that is identical except for thechange. Alternatively, the change may increase the melting point by morethan 0.5° C. or even 1.0° C. or more. A method for determining themelting point of Fn3 molecules is given in Example 19 below.

[0023] The Fn3 may have at least one aspartic acid (Asp) residue and/orat least one glutamic acid (Glu) residue that has been deleted orsubstituted with at least one other amino acid residue. For example, Asp7 and/or Asp 23 and/or Glu 9, may have been deleted or substituted withat least one other amino acid residue. Asp 7, Asp 23, or Glu 9, may havebeen substituted with an asparagine (Asn) or lysine (Lys) residue. Thepresent invention further provides an isolated nucleic acid molecule andan expression vector encoding an Fn3 molecule wherein the Fn3 contains astabilizing mutation.

[0024] The invention provides a fibronectin type III (Fn3) polypeptidemonobody containing a plurality of Fn3 β-strand domain sequences thatare linked to a plurality of loop region sequences wherein the Fn3contains a stabilizing mutation. One or more of the monobody loop regionsequences of the Fn3 polypeptide vary by deletion, insertion orreplacement of at least two amino acids from the corresponding loopregion sequences in wild-type Fn3. The β-strand domains of the monobodyhave at least about 50% total amino acid sequence homology to thecorresponding amino acid sequence of wild-type Fn3's β-strand domainsequences. Preferably, one or more of the loop regions of the monobodycontain amino acid residues:

[0025] i) from 15 to 16 inclusive in an AB loop;

[0026] ii) from 22 to 30 inclusive in a BC loop;

[0027] iii) from 39 to 45 inclusive in a CD loop;

[0028] iv) from 51 to 55 inclusive in a DE loop;

[0029] v) from 60 to 66 inclusive in an EF loop; and

[0030] vi) from 76 to 87 inclusive in an FG loop.

[0031] The invention also provides a nucleic acid molecule encoding aFn3 polypeptide monobody wherein the Fn3 contains a stabilizingmutation, as well as an expression vector containing the nucleic acidmolecule and a host cell containing the vector.

[0032] The invention further provides a method of preparing a Fn3polypeptide monobody wherein the Fn3 contains a stabilizing mutation.The method includes providing a DNA sequence encoding a plurality of Fn3β-strand domain sequences that are linked to a plurality of loop regionsequences, wherein at least one loop region of the sequence contains aunique restriction enzyme site. The DNA sequence is cleaved at theunique restriction site. Then a preselected DNA segment is inserted intothe restriction site. The preselected DNA segment encodes a peptidecapable of binding to a specific binding partner (SBP) or a transitionstate analog compound (TSAC). The insertion of the preselected DNAsegment into the DNA sequence yields a DNA molecule which encodes apolypeptide monobody having an insertion. The DNA molecule is thenexpressed so as to yield the polypeptide monobody.

[0033] Also provided is a method of preparing a Fn3 polypeptide monobodywherein the Fn3 contains a stabilizing mutation, which method includesproviding a replicatable DNA sequence encoding a plurality of Fn3β-strand domain sequences that are linked to a plurality of loop regionsequences, wherein the nucleotide sequence of at least one loop regionis known. Polymerase chain reaction (PCR) primers are provided orprepared which are sufficiently complementary to the known loop sequenceso as to be hybridizable under PCR conditions, wherein at least one ofthe primers contains a modified nucleic acid sequence to be insertedinto the DNA sequence. PCR is performed using the replicatable DNAsequence and the primers. The reaction product of the PCR is thenexpressed so as to yield a polypeptide monobody.

[0034] The invention provides a further method of preparing a Fn3polypeptide monobody wherein the Fn3 contains a stabilizing mutation.The method includes providing a replicatable DNA sequence encoding aplurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein the nucleotide sequence ofat least one loop region is known. Site-directed mutagenesis of at leastone loop region is performed so as to create an insertion mutation. Theresultant DNA including the insertion mutation is then expressed.

[0035] Further provided is a variegated nucleic acid library encodingFn3 polypeptide monobodies including a plurality of nucleic acid speciesencoding a plurality of Fn3 β-strand domain sequences that are linked toa plurality of loop region sequences, wherein one or more of themonobody loop region sequences vary by deletion, insertion orreplacement of at least two amino acids from corresponding loop regionsequences in wild-type Fn3, and wherein the β-strand domains of themonobody have at least a 50% total amino acid sequence homology to thecorresponding amino acid sequence of β-strand domain sequences of thewild-type Fn3, and wherein the Fn3 contains a stabilizing mutation. Theinvention also provides a peptide display library derived from thevariegated nucleic acid library of the invention. Preferably, thepeptide of the peptide display library is displayed on the surface of abacteriophage, e.g., a M13 bacteriophage or a fd bacteriophage, orvirus.

[0036] The invention also provides a method of identifying the aminoacid sequence of a polypeptide molecule capable of binding to a specificbinding partner (SBP) so as to form a polypeptide:SSP complex, whereinthe dissociation constant of the the polypeptide:SBP complex is lessthan 10⁻⁶ moles/liter. The method includes the steps of:

[0037] a) providing a peptide display library of the invention;

[0038] b) contacting the peptide display library of (a) with animmobilized or separable SBP;

[0039] c) separating the peptide:SBP complexes from the free peptides;

[0040] d) causing the replication of the separated peptides of (c) so asto result in a new peptide display library distinguished from that in(a) by having a lowered diversity and by being enriched in displayedpeptides capable of binding the SBP;

[0041] e) optionally repeating steps (b), (c), and (d) with the newlibrary of (d); and

[0042] f) determining the nucleic acid sequence of the region encodingthe displayed peptide of a species from (d) and hence deducing thepeptide sequence capable of binding to the SBP.

[0043] The present invention also provides a method of preparing avariegated nucleic acid library encoding Fn3 polypeptide monobodieshaving a plurality of nucleic acid species each including a plurality ofloop regions, wherein the species encode a plurality of Fn3 β-stranddomain sequences that are linked to a plurality of loop regionsequences, wherein one or more of the loop region sequences vary bydeletion, insertion or replacement of at least two amino acids fromcorresponding loop region sequences in wild-type Fn3, and wherein theβ-strand domain sequences of the monobody have at least a 50% totalamino acid sequence homology to the corresponding amino acid sequencesof β-strand domain sequences of the wild-type Fn3, and wherein the Fn3contains a stabilizing mutation, including the steps of

[0044] a) preparing an Fn3 polypeptide monobody having a predeterminedsequence;

[0045] b) contacting the polypeptide with a specific binding partner(SBP) so as to form a polypeptide:SSP complex wherein the dissociationconstant of the the polypeptide:SBP complex is less than 10⁻⁶moles/liter;

[0046] c) determining the binding structure of the polypeptide:SBPcomplex by nuclear magnetic resonance spectroscopy or X-raycrystallography; and

[0047] d) preparing the variegated nucleic acid library, wherein thevariegation is performed at positions in the nucleic acid sequencewhich, from the information provided in (c), result in one or morepolypeptides with improved binding to the SBP.

[0048] Also provided is a method of identifying the amino acid sequenceof a polypeptide molecule capable of catalyzing a chemical reaction witha catalyzed rate constant, k_(cat), and an uncatalyzed rate constant,k_(uncat), such that the ratio of k_(cat)/k_(uncat) is greater than 10.The method includes the steps of:

[0049] a) providing a peptide display library of the invention;

[0050] b) contacting the peptide display library of (a) with animmobilized or separable transition state analog compound (TSAC)representing the approximate molecular transition state of the chemicalreaction;

[0051] c) separating the peptide:TSAC complexes from the free peptides;

[0052] d) causing the replication of the separated peptides of (c) so asto result in a new peptide display library distinguished from that in(a) by having a lowered diversity and by being enriched in displayedpeptides capable of binding the TSAC;

[0053] e) optionally repeating steps (b), (c), and (d) with the newlibrary of (d); and

[0054] f) determining the nucleic acid sequence of the region encodingthe displayed peptide of a species from (d) and hence deducing thepeptide sequence.

[0055] The invention also provides a method of preparing a variegatednucleic acid library encoding Fn3 polypeptide monobodies having aplurality of nucleic acid species each including a plurality of loopregions, wherein the species encode a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein one or more of the loop region sequences vary by deletion,insertion or replacement of at least two amino acids from correspondingloop region sequences in wild-type Fn3, and wherein the β-strand domainsequences of the monobody have at least a 50% total amino acid sequencehomology to the corresponding amino acid sequences of β-strand domainsequences of the wild-type Fn3, and wherein the Fn3 contains astabilizing mutation, including the steps of

[0056] a) preparing an Fn3 polypeptide monobody having a predeterminedsequence, wherein the polypeptide is capable of catalyzing a chemicalreaction with a catalyzed rate constant, k_(cat), and an uncatalyzedrate constant, k_(uncat), such that the ratio of k_(cat)/K_(uncat) isgreater than 10;

[0057] b) contacting the polypeptide with an immobilized or separabletransition state analog compound (TSAC) representing the approximatemolecular transition state of the chemical reaction;

[0058] c) determining the binding structure of the polypeptide:TSACcomplex by nuclear magnetic resonance spectroscopy or X-raycrystallography; and

[0059] d) preparing the variegated nucleic acid library, wherein thevariegation is performed at positions in the nucleic acid sequencewhich, from the information provided in (c), result in one or morepolypeptides with improved binding to or stabilization of the TSAC.

[0060] The invention also provides a kit for the performance of any ofthe methods of the invention. The invention further provides acomposition, e.g., a polypeptide, prepared by the use of the kit, oridentified by any of the methods of the invention.

[0061] The following abbreviations have been used in describing aminoacids, peptides, or proteins: Ala or A, Alanine; Arg or R, Arginine; Asnor N asparagine; Asp or D, aspartic acid; Cys or C, cysteine; Gln or Q,glutamine; Glu or E, glutamic acid; Gly or G, glycine; His or H,histidine; Ile or I, isoleucine; Leu or L, leucine; Lys or K, lysine;Met or M, methionine; Phe or F, phenylalanine; Pro or P, proline; Ser orS, serine; Thr or T, threonine; Trp or W, tryptophan; Tyr or Y,tyrosine; Val or V, valine.

[0062] The following abbreviations have been used in describing nucleicacids, DNA, or RNA: A, adenosine; T, thymidine; G, guanosine; C,cytosine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063]FIG. 1. β-Strand and loop topology (A, B) and MOLSCRIPTrepresentation (C, D; Kraulis, 1991) of the VH domain of anti-lysozymeimmunoglobulin D1.3 (A, C; Bhat et al., 1994) and 10th type III domainof human fibronectin (B, D; Main et al., 1992). The locations ofcomplementarity determining regions (CDRs, hypervariable regions) andthe integrin-binding Arg-Gly-Asp (RGD) sequence are indicated.

[0064]FIG. 2. Amino acid sequence (SEQ ID NO: 110) and restriction sitesof the synthetic Fn3 gene. The residue numbering is according to Main etal. (1992). Restriction enzyme sites designed are shown above the aminoacid sequence. β-Strands are denoted by underlines. The N-terminal “mq”sequence has been added for a subsequent cloning into an expressionvector. The His.tag (Novagen) fusion protein has an additional sequence,MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 114), preceding the Fn3 sequence shownabove.

[0065]FIG. 3. A, Far UV CD spectra of wild-type Fn3 at 25° C. and 90° C.Fn3 (50 μM) was dissolved in sodium acetate (50 mM, pH 4.6). B, thermaldenaturation of Fn3 monitored at 215 nm. Temperature was increased at arate of 1° C./min.

[0066]FIG. 4. A, Cα trace of the crystal structure of the complex oflysozyme (HEL) and the Fv fragment of the anti-hen egg-white lysozyme(anti-HEL) antibody D1.3 (Bhat et al., 1994). Side chains of theresidues 99-102 of VH CDR3, which make contact with HEL, are also shown.B, Contact surface area for each residue of the D1.3 VH-HEL and VH-VLinteractions plotted vs. residue number of D1.3 VH. Surface area andsecondary structure were determined using the program DSSP (Kabsh andSander, 1983). C and D, schematic drawings of the β-sheet structure ofthe F strand-loop-G strand moieties of D1.3 VH (C) and Fn3 (D). Theboxes denote residues in β-strands and ovals those not in strands. Theshaded boxes indicate residues of which side chains are significantlyburied. The broken lines indicate hydrogen bonds.

[0067]FIG. 5. Designed Fn3 gene showing DNA (SEQ ID NO:111) and aminoacid (SEQ ID NO: 112) sequences. The amino acid numbering is accordingto Main et al. (1992). The two loops that were randomized incombinatorial libraries are enclosed in boxes.

[0068]FIG. 6. Map of plasmid pAS45. Plasmid pAS45 is the expressionvector of His.tag-Fn3.

[0069]FIG. 7. Map of plasmid pAS25. Plasmid pAS25 is the expressionvector of Fn3.

[0070]FIG. 8. Map of plasmid pAS38. pAS38 is a phagmid vector for thesurface display of Fn3.

[0071]FIG. 9. (Ubiquitin-1) Characterization of ligand-specific bindingof enriched clones using phage enzyme-linked immunosolvent assay(ELISA). Microtiter plate wells were coated with ubiquitin (1 μg/well;“Ligand (+)) and then blocked with BSA. Phage solution in TBS containingapproximately 10¹⁰ colony forming units (cfu) was added to a well andwashed with TBS. Bound phages were detected with anti-phage antibody-PODconjugate (Pharmacia) with Turbo-TMB (Pierce) as a substrate. Absorbancewas measured using a Molecular Devices SPECTRAmax 250 microplatespectrophotometer. For a control, wells without the immobilized ligandwere used. 2-1 and 2-2 denote enriched clones from Library 2 eluted withfree ligand and acid, respectively. 4-1 and 4-2 denote enriched clonesfrom Library 4 eluted with free ligand and acid, respectively.

[0072]FIG. 10. (Ubiquitin-2) Competition phage ELISA of enriched clones.Phage solutions containing approximately 10¹⁰ cfu were first incubatedwith free ubiquitin at 4° C. for 1 hour prior to the binding to aligand-coated well. The wells were washed and phages detected asdescribed above.

[0073]FIG. 11. Competition phage ELISA of ubiquitin-binding monobody411. Experimental conditions are the same as described above forubiquitin. The ELISA was performed in the presence of free ubiquitin inthe binding solution. The experiments were performed with four differentpreparations of the same clone.

[0074]FIG. 12. (Fluorescein-1) Phage ELISA of four clones, Plb25.1(containing SEQ ID NO:115), Plb25.4 (containing SEQ ID NO:116), pLB24.1(containing SEQ ID NO:117) and pLB24.3 (containing SEQ ID NO:118).Experimental conditions are the same as ubiquitin-1 above.

[0075]FIG. 13. (Fluorescein-2) Competition ELISA of the four clones.Experimental conditions are the same as ubiquitin-2 above.

[0076]FIG. 14. ¹H, ¹⁵N-HSQC spectrum of a fluorescence-binding monobodyLB25.5. Approximately 20 μM protein was dissolved in 10 mM sodiumacetate buffer (pH 5.0) containing 100 mM sodium chloride. The spectrumwas collected at 30° C. on a Varian Unity INOVA 600 NMR spectrometer.

[0077]FIG. 15. Characterization of the binding reaction of Ubi4-Fn3 tothe target, ubiquitin. (a) Phage ELISA analysis of binding of Ubi4-Fn3to ubiquitin. The binding of Ubi4-phages to ubiquitin-coated wells wasmeasured. The control experiment was performed with wells containing noubiquitin.

[0078] (b) Competition phage ELISA of Ubi4-Fn3. Ubi4-Fn3-phages werepreincubated with soluble ubiquitin at an indicated concentration,followed by the phage ELISA detection in ubiquitin-coated wells.

[0079] (c) Competition phage ELISA testing the specificity of the Ubi4clone. The Ubi4 phages were preincubated with 250 μg/ml of solubleproteins, followed by phage ELISA as in (b).

[0080] (d) ELISA using free proteins.

[0081]FIG. 16. Equilibrium unfolding curves for Ubi4-Fn3 (closedsymbols) and wild-type Fn3 (open symbols). Squares indicate datameasured in TBS (Tris HCl buffer (50 mM, pH 7.5) containing NaCl (150mM)). Circles indicate data measured in Gly HCl buffer (20 mM, pH 3.3)containing NaCl (300 mM). The curves show the best fit of the transitioncurve based on the two-state model. Parameters characterizing thetransitions are listed in Table 8.

[0082]FIG. 17. (a) ¹H, ¹⁵N-HSQC spectrum of [¹⁵N]-Ubi4-K Fn3. (b).Difference (δ_(wild-type)−δ_(Ubi4)) of ¹H (b) and ¹⁵N (c) chemicalshifts plotted versus residue number. Values for residues 82-84 (shownas filled circles) where Ubi4-K deletions are set to zero. Open circlesindicate residues that are mutated in the Ubi4-K protein. The locationsof β-strands are indicated with arrows.

[0083]FIG. 18. (A) Guanidine hydrochloride (GuHCl)-induced denaturationof FNfn10 monitored by Trp fluorescence. The fluorescence emissionintensity at 355 nm is shown as a function of GuHCl concentration. Thelines show the best fits of the data to the two-state transition model.(B) Stability of FN3 at 4 M GuHCl plotted as a function of pH. (C) pHdependence of the m value.

[0084]FIG. 19. A two-dimensional H(C)CO spectrum of FNfn10 showing the¹³C chemical shift of the carboxyl carbon (vertical axis) and the ¹Hshift of ¹H^(β) of Asp or ¹H^(γ) of Glu, respectively (horizontal axis).Cross peaks are labeled with their respective residue numbers.

[0085]FIG. 20. pH-Dependent shifts of the ¹³C chemical shifts of thecarboxyl carbons of Asp and Glu residues in FNfn10. Panel A shows datafor Asp 3, 67 and 80, and Glu 38 and 47. The lines are the best fits ofthe data to the Henderson-Hasselbalch equation with one ionizable group(McIntosh, L. P., Hand, G., Johnson, P. E., Joshi, M. D., Koerner, M.,Plesniak, L. A., Ziser, L., Wakarchuk, W. W. & Withers, S. G. (1996)Biochemistry 35, 9958-9966). Panel B shows data for Asp 7 and 23 and Glu9. The continuous lines show the best fits to the Henderson-Hasselbalchequation with two ionizable groups, while the dashed lines show the bestfits to the equation with a single ionizable group.

[0086]FIG. 21. (A) The amino acid sequence of FNfn10 (SEQ ID NO:121)shown according to its topology (Main, A. L., Harvey, T. S., Baron, M.,Boyd, J., & Campbell, I. D. (1992) Cell 71, 671-678). Asp and Gluresidues are highlighted with gray circles. The thin lines and arrowsconnecting circles indicate backbone hydrogen bonds. (B) A CPK model ofFN3 showing the locations of Asp 7 and 23 and Glu 9.

[0087]FIG. 22. Thermal denaturation of the wild-type and mutant FNfn10proteins at pH 7.0 and 2.4 in the presence of 6.3 M urea and 0.1 or 1.0M NaCl. Change in circular dichroism signal at 227 nm is plotted as afunction of temperature. The filled circles show the data in thepresence of 1 M NaCl and the open circles are data in the presence of0.1 M NaCl. The left column shows data taken at pH 2.4 and the rightcolumn at pH 7.0. The identity of proteins is indicated in the panels.

[0088]FIG. 23. GuHCl-induce denaturation of FNfn10 mutants monitoredwith fluorescence. Fluorescence data was converted to the fraction ofunfolded protein according to the two-state transition model (Loladze,V. V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999)Biochemistry 38, 16419-16423), and plotted as a function of GuHCl.

[0089]FIG. 24. pH Titration of the carboxyl ¹³C resonance of Asp and Gluresidues in D7N (open circles) and D7K (closed circles) FNfn10. Data forthe wild-type (crosses) are also shown for comparison. Residue names aredenoted in the individual panels.

DETAILED DESCRIPTION OF THE INVENTION

[0090] For the past decade the immune system has been exploited as arich source of de novo catalysts. Catalytic antibodies have been shownto have chemoselectivity, enantioselectivity, large rate accelerations,and even an ability to reroute chemical reactions. In most cases theantibodies have been elicited to transition state analog (TSA) haptens.These TSA haptens are stable, low-molecular weight compounds designed tomimic the structures of the energetically unstable transition statespecies that briefly (approximate half-life 10¹³ s) appear alongreaction pathways between reactants and products. Anti-TSA antibodies,like natural enzymes, are thought to selectively bind and stabilizetransition state, thereby easing the passage of reactants to products.Thus, upon binding, the antibody lowers the energy of the actualtransition state and increases the rate of the reaction. These catalystscan be programmed to bind to geometrical and electrostatic features ofthe transition state so that the reaction route can be controlled byneutralizing unfavorable charges, overcoming entropic barriers, anddictating stereoelectronic features of the reaction. By this means evenreactions that are otherwise highly disfavored have been catalyzed(Janda et al. 1997). Further, in many instances catalysts have been madefor reactions for which there are no known natural or man-made enzymes.

[0091] The success of any combinatorial chemical system in obtaining aparticular function depends on the size of the library and the abilityto access its members. Most often the antibodies that are made in ananimal against a hapten that mimics the transition state of a reactionare first screened for binding to the hapten and then screened again forcatalytic activity. An improved method allows for the direct selectionfor catalysis from antibody libraries in phage, thereby linkingchemistry and replication.

[0092] A library of antibody fragments can be created on the surface offilamentous phage viruses by adding randomized antibody genes to thegene that encodes the phage's coat protein. Each phage then expressesand displays multiple copies of a single antibody fragment on itssurface. Because each phage possesses both the surface-displayedantibody fragment and the DNA that encodes that fragment, and antibodyfragment that binds to a target can be identified by amplifying theassociated DNA.

[0093] Immunochemists use as antigens materials that have as littlechemical reactivity as possible. It is almost always the case that onewishes the ultimate antibody to interact with native structures. Inreactive immunization the concept is just the opposite. One immunizeswith compounds that are highly reactive so that upon binding to theantibody molecule during the induction process, a chemical reactionensues. Later this same chemical reaction becomes part of the mechanismof the catalytic event. In a certain sense one is immunizing with achemical reaction rather than a substance per se. Reactive immunogenscan be considered as analogous to the mechanism-based inhibitors thatenzymologists use except that they are used in the inverse way in that,instead of inhibiting a mechanism, they induce a mechanism.

[0094] Man-made catalytic antibodies have considerable commercialpotential in many different applications. Catalytic antibody-basedproducts have been used successfully in prototype experiments intherapeutic applications, such as prodrug activation and cocaineinactivation, and in nontherapeutic applications, such as biosensors andorganic synthesis.

[0095] Catalytic antibodies are theoretically more attractive thannoncatalytic antibodies as therapeutic agents because, being catalytic,they may be used in lower doses, and also because their effects areunusually irreversible (for example, peptide bond cleavage rather thanbinding). In therapy, purified catalytic antibodies could be directlyadministered to a patient, or alternatively the patient's own catalyticantibody response could be elicited by immunization with an appropriatehapten. Catalytic antibodies also could be used as clinical diagnostictools or as regioselective or stereoselective catalysts in the synthesisof fine chemicals.

[0096] I. Mutation of Fn3 loops and grafting of Ab loops onto Fn3

[0097] An ideal scaffold for CDR grafting is highly soluble and stable.It is small enough for structural analysis, yet large enough toaccommodate multiple CDRs so as to achieve tight binding and/or highspecificity.

[0098] A novel strategy to generate an artificial Ab system on theframework of an existing non-Ab protein was developed. An advantage ofthis approach over the minimization of an Ab scaffold is that one canavoid inheriting the undesired properties of Abs. Fibronectin type IIIdomain (Fn3) was used as the scaffold. Fibronectin is a large proteinwhich plays essential roles in the formation of extracellular matrix andcell-cell interactions; it consists of many repeats of three types (I,II and III) of small domains (Baron et al., 1991). Fn3 itself is theparadigm of a large subfamily (Fn3 family or s-type Ig family) of theimmunoglobulin superfamily (IgSF). The Fn3 family includes cell adhesionmolecules, cell surface hormone and cytokine receptors, chaperoning, andcarbohydrate-binding domains (for reviews, see Bork & Doolittle, 1992;Jones, 1993; Bork et al., 1994; Campbell & Spitzfaden, 1994; Harpez &Chothia, 1994).

[0099] Recently, crystallographic studies revealed that the structure ofthe DNA binding domains of the transcription factor NF-kB is alsoclosely related to the Fn3 fold (Ghosh et al., 1995; Müller et al.,1995). These proteins are all involved in specific molecularrecognition, and in most cases ligand-binding sites are formed bysurface loops, suggesting that the Fn3 scaffold is an excellentframework for building specific binding proteins. The 3D structure ofFn3 has been determined by NMR (Main et al., 1992) and by X-raycrystallography (Leahy et al., 1992; Dickinson et al., 1994). Thestructure is best described as a β-sandwich similar to that of Ab VHdomain except that Fn3 has seven β-strands instead of nine (FIG. 1).There are three loops on each end of Fn3; the positions of the BC, DEand FG loops approximately correspond to those of CDR1, 2 and 3 of theVH domain, respectively (FIG. 1 C, D).

[0100] Fn3 is small (˜95 residues), monomeric, soluble and stable. It isone of few members of IgSF that do not have disulfide bonds; VH has aninterstrand disulfide bond (FIG. 1 A) and has marginal stability underreducing conditions. Fn3 has been expressed in E. coli (Aukhil et al.,1993). In addition, 17 Fn3 domains are present just in humanfibronectin, providing important information on conserved residues whichare often important for the stability and folding (for sequencealignment, see Main et al., 1992 and Dickinson et al., 1994). Fromsequence analysis, large variations are seen in the BC and FG loops,suggesting that the loops are not crucial to stability. NMR studies haverevealed that the FG loop is highly flexible; the flexibility has beenimplicated for the specific binding of the 10th Fn3 to α₅β₁ integrinthrough the Arg-Gly-Asp (RGD) motif. In the crystal structure of humangrowth hormone-receptor complex (de Vos et al., 1992), the second Fn3domain of the receptor interacts with hormone via the FG and BC loops,suggesting it is feasible to build a binding site using the two loops.

[0101] The tenth type III module of fibronectin has a fold similar tothat of immunoglobulin domains, with seven β strands forming twoantiparallel β sheets, which pack against each other (Main et al.,1992). The structure of the type II module consists of seven β strands,which form a sandwich of two antiparallel β sheets, one containing threestrands (ABE) and the other four strands (C′CFG) (Williams et al.,1988). The triple-stranded β sheet consists of residues Glu-9-Thr-14(A), Ser-17-Asp-23 (B), and Thr-56-Ser-60 (E). The majority of theconserved residues contribute to the hydrophobic core, with theinvariant hydrophobic residues Trp-22 and Try-68 lying toward theN-terminal and C-terminal ends of the core, respectively. The β strandsare much less flexible and appear to provide a rigid framework uponwhich functional, flexible loops are built. The topology is similar tothat of immunoglobulin C domains.

[0102] Gene construction and mutagenesis

[0103] A synthetic gene for tenth Fn3 of human fibronectin (FIG. 2) wasdesigned which includes convenient restriction sites for ease ofmutagenesis and uses specific codons for high-level protein expression(Gribskov et al., 1984).

[0104] The gene was assembled as follows: (1) the gene sequence wasdivided into five parts with boundaries at designed restriction sites(FIG. 2); (2) for each part, a pair of oligonucleotides that codeopposite strands and have complementary overlaps of˜15 bases wassynthesized; (3) the two oligonucleotides were annealed and singlestrand regions were filled in using the Klenow fragment of DNApolymerase; (4) the double-stranded oligonucleotide was cloned into thepET3a vector (Novagen) using restriction enzyme sites at the termini ofthe fragment and its sequence was confirmed by an Applied Biosystems DNAsequencer using the dideoxy termination protocol provided by themanufacturer; (5) steps 2-4 were repeated to obtain the whole gene(plasmid pAS25) (FIG. 7).

[0105] Although the present method takes more time to assemble a genethan the one-step polymerase chain reaction (PCR) method (Sandhu et al.,1992), no mutations occurred in the gene. Mutations would likely havebeen introduced by the low fidelity replication by Taq polymerase andwould have required time-consuming gene editing. The gene was alsocloned into the pET15b (Novagen) vector (pEW1). Both vectors expressedthe Fn3 gene under the control of bacteriophage T7 promoter (Studler etal. 1990); pAS25 expressed the 96-residue Fn3 protein only, while pEW1expressed Fn3 as a fusion protein with poly-histidine peptide (His.tag).Recombinant DNA manipulations were performed according to MolecularCloning (Sambrook et al., 1989), unless otherwise stated.

[0106] Mutations were introduced to the Fn3 gene using either cassettemutagenesis or oligonucleotide site-directed mutagenesis techniques(Deng & Nickoloff, 1992). Cassette mutagenesis was performed using thesame protocol for gene construction described above; double-stranded DNAfragment coding a new sequence was cloned into an expression vector(pAS25 and/or pEW1). Many mutations can be made by combining a newlysynthesized strand (coding mutations) and an oligonucleotide used forthe gene synthesis. The resulting genes were sequenced to confirm thatthe designed mutations and no other mutations were introduced bymutagenesis reactions.

[0107] Design and synthesis of Fn3 mutants with antibody CDRs

[0108] Two candidate loops (FG and BC) were identified for grafting.Antibodies with known crystal structures were examined in order toidentify candidates for the sources of loops to be grafted onto Fn3.Anti-hen egg lysozyme (HEL) antibody D1.3 (Bhat et al., 1994) was chosenas the source of a CDR loop. The reasons for this choice were: (1) highresolution crystal structures of the free and complexed states areavailable (FIG. 4 A; Bhat et al., 1994), (2) thermodynamics data for thebinding reaction are available (Tello et al., 1993), (3) D1.3 has beenused as a paradigm for Ab structural analysis and Ab engineering(Verhoeyen et al., 1988; McCafferty et al., 1990) (4) site-directedmutagenesis experiments have shown that CDR3 of the heavy chain(VH-CDR3) makes a larger contribution to the affinity than the otherCDRs (Hawkins et al., 1993), and (5) a binding assay can be easilyperformed. The objective for this trial was to graft VH-CDR3 of D1.3onto the Fn3 scaffold without significant loss of stability.

[0109] An analysis of the D1.3 structure (FIG. 4) revealed that onlyresidues 99-102 (“RDYR”) (SEQ ID NO:120) make direct contact with henegg-white lysozyme (HEL) (FIG. 4 B), although VH-CDR3 is defined aslonger (Bhat et al., 1994). It should be noted that the C-terminal halfof VH-CDR3 (residues 101-104) made significant contact with the VLdomain (FIG. 4 B). It has also become clear that D1.3 VH-CDR3 (FIG. 4 C)has a shorter turn between the strands F and G than the FG loop of Fn3(FIG. 4 D). Therefore, mutant sequences were designed by using the RDYR(99-102) (SEQ ID NO: 120) of D1.3 as the core and made differentboundaries and loop lengths (Table 1). Shorter loops may mimic the D1.3CDR3 conformation better, thereby yielding higher affinity, but they mayalso significantly reduce stability by removing wild-type interactionsof Fn3. TABLE 1 Amino acid sequences of D1.3 VH CDR3, VH8 CDR3 and Fn3FG loop and list of planned mutants. 96     100         105•       •           • D1.3 A R E R D Y R L D Y W G Q G (SEQ ID NO:1) VH8A R G A V V S Y Y A M D Y W G Q G (SEQ ID NO:2)     75        80        85       •         •         • Fn3Y A V T G R G D S P A S S K P I (SEQ ID NO:3) Mutant Sequence D1.3-1Y A E R D Y R L D Y - - - - P I (SEQ ID NO:4) D1.3-2Y A V R D Y R L D Y - - - - P I (SEQ ID NO:5) D1.3-3Y A V R D Y R L D Y A S S K P I (SEQ ID NO:6) D1.3-4Y A V R D Y R L D Y - - - K P I (SEQ ID NO:7) D1.3-5Y A V R D Y R - - - - - S K P I (SEQ ID NO:8) D1.3-6Y A V T R D Y R L - - S S K P I (SEQ ID NO:9) D1.3-7Y A V T E R D Y R L - S S K P I (SEQ ID NO:10) VH8-1Y A V A V V S Y Y A M D Y - P I (SEQ ID NO:11) VH8-2Y A V T A V V S Y Y A S S K P I (SEQ ID NO:12)

[0110] In addition, an anti-HEL single VH domain termed VH8 (Ward etal., 1989) was chosen as a template. VH8 was selected by libraryscreening and, in spite of the lack of the VL domain, VH8 has anaffinity for HEL of 27 nM, probably due to its longer VH-CDR3 (Table 1).Therefore, its VH-CDR3 was grafted onto Fn3. Longer loops may beadvantageous on the Fn3 framework because they may provide higheraffinity and also are close to the loop length of wild-type Fn3. The 3Dstructure of VH8 was not known and thus the VH8 CDR3 sequence wasaligned with that of D1.3 VH-CDR3; two loops were designed (Table 1).

[0111] Mutant construction and production

[0112] Site-directed mutagenesis experiments were performed to obtaindesigned sequences. Two mutant Fn3s, D1.3-1 and D1.3-4 (Table 1) wereobtained and both were expressed as soluble His.tag fusion proteins.D1.3-4 was purified and the His.tag portion was removed by thrombincleavage. D1.3-4 is soluble up to at least 1 mM at pH 7.2. Noaggregation of the protein has been observed during sample preparationand NMR data acquisition.

[0113] Protein expression and purification

[0114]E. coli BL21 (DE3) (Novagen) were transformed with an expressionvector (pAS25, pEW1 and their derivatives) containing a gene for thewild-type or a mutant. Cells were grown in M9 minimal medium and M9medium supplemented with Bactotrypton (Difco) containing ampicillin (200μg/ml). For isotopic labeling, ¹⁵N NH₄Cl and/or ¹³C glucose replacedunlabeled components. 500 ml medium in a 2 liter baffle flask wereinoculated with 10 ml of overnight culture and agitated at 37° C.Isopropylthio-β-galactoside (IPTG) was added at a final concentration of1 mM to initiate protein expression when OD (600 nm) reaches one. Thecells were harvested by centrifugation 3 hours after the addition ofIPTG and kept frozen at −70° C. until used.

[0115] Fn3 without His.tag was purified as follows. Cells were suspendedin 5 ml/(g cell) of Tris (50 mM, pH 7.6) containingethylenediaminetetraacetic acid (EDTA; 1 mM) and phenylmethylsulfonylfluoride (1 mM). HEL was added to a final concentration of 0.5 mg/ml.After incubating the solution for 30 minutes at 37° C., it was sonicatedthree times for 30 seconds on ice. Cell debris was removed bycentrifugation. Ammonium sulfate was added to the solution andprecipitate recovered by centrifugation. The pellet was dissolved in5-10 ml sodium acetate (50 mM, pH 4.6) and insoluble material wasremoved by centrifugation. The solution was applied to a SephacrylS100HR column (Pharmacia) equilibrated in the sodium acetate buffer.Fractions containing Fn3 then was applied to a ResourceS column(Pharmacia) equilibrated in sodium acetate (50 mM, pH 4.6) and elutedwith a linear gradient of sodium chloride (0-0.5 M). The protocol can beadjusted to purify mutant proteins with different surface chargeproperties.

[0116] Fn3 with His.tag was purified as follows. The soluble fractionwas prepared as described above, except that sodium phosphate buffer (50mM, pH 7.6) containing sodium chloride (100 mM) replaced the Trisbuffer. The solution was applied to a Hi-Trap chelating column(Pharmacia) preloaded with nickel and equilibrated in the phosphatebuffer. After washing the column with the buffer, His.tag-Fn3 was elutedin the phosphate buffer containing 50 mM EDTA. Fractions containingHis.tag-Fn3 were pooled and applied to a Sephacryl S100-HR column,yielding highly pure protein. The His.tag portion was cleaved off bytreating the fusion protein with thrombin using the protocol supplied byNovagen. Fn3 was separated from the His.tag peptide and thrombin by aResourceS column using the protocol above.

[0117] The wild-type and two mutant proteins so far examined areexpressed as soluble proteins. In the case that a mutant is expressed asinclusion bodies (insoluble aggregate), it is first examined if it canbe expressed as a soluble protein at lower temperature (e.g., 25-30°C.). If this is not possible, the inclusion bodies are collected bylow-speed centrifugation following cell lysis as described above. Thepellet is washed with buffer, sonicated and centrifuged. The inclusionbodies are solubilized in phosphate buffer (50 mM, pH 7.6) containingguanidinium chloride (GdnCl, 6 M) and will be loaded on a Hi-Trapchelating column. The protein is eluted with the buffer containing GdnCland 50 mM EDTA.

[0118] Conformation of mutant Fn3, D1.3-4

[0119] The ¹H NMR spectra of His.tag D1.3-4 fusion protein closelyresembled that of the wild-type, suggesting the mutant is folded in asimilar conformation to that of the wild-type. The spectrum of D1.3-4after the removal of the His.tag peptide showed a large spectraldispersion. A large dispersion of amide protons (7-9.5 ppm) and a largenumber of downfield (5.0-6.5 ppm) C^(α) protons are characteristic of aβ-sheet protein (Wüthrich, 1986).

[0120] The 2D NOESY spectrum of D1.3-4 provided further evidence for apreserved conformation. The region in the spectrum showed interactionsbetween upfield methyl protons (<0.5 ppm) and methyl-methylene protons.The Val72 γ methyl resonances were well separated in the wild-typespectrum (−0.07 and 0.37 ppm; (Baron et al., 1992)). Resonancescorresponding to the two methyl protons are present in the D1.3-4spectrum (−0.07 and 0.44 ppm). The cross peak between these tworesonances and other conserved cross peaks indicate that the tworesonances in the D1.3-4 spectrum are highly likely those of Val72 andthat other methyl protons are in nearly identical environment to that ofwild-type Fn3. Minor differences between the two spectra are presumablydue to small structural perturbation due to the mutations. Val72 is onthe F strand, where it forms a part of the central hydrophobic core ofFn3 (Main et al., 1992). It is only four residues away from the mutatedresidues of the FG loop (Table 1). The results are remarkable because,despite there being 7 mutations and 3 deletions in the loop (more than10% of total residues; FIG. 12, Table 2), D1.3-4 retains a 3D structurevirtually identical to that of the wild-type (except for the mutatedloop). Therefore, the results provide strong support that the FG loop isnot significantly contributing to the folding and stability of the Fn3molecule and thus that the FG loop can be mutated extensively. TABLE 2Sequences of oligonucleotides Name Sequence FN1FCGGGATCCCATATGCAGGTTTCTG (SEQ ID NO:13) ATGTTCCGCGTGACCTGGAAGTTGTTGCTGCGACC FN1R TAACTGCAGGAGCATCCCAGCTGA (SEQ ID NO:14)TCAGCAGGCTAGTCGGGGTCGCAG CAACAAC FN2F CTCCTGCAGTTACCGTGCGTTATT (SEQ IDNO:15) ACCGTATCACGTACGGTGAAACCG GTG FN2R GTGAATTCCTGAACCGGGGAGTTA (SEQID NO:16) CCACCGGTTTCACCG FN3F AGGAATTCACTGTACCTGGTTCCA (SEQ ID NO:17)AGTCTACTGCTACCATCAGCGG FN3R GTATAGTCGACACCCGGTTTCAGG (SEQ ID NO:18)CCGCTGATGGTAGC FN4F CGGGTGTCGACTATACCATCACTG (SEQ ID NO: 19) TATACGCTFN4R CGGGATCCGAGCTCGCTGGGCTGT (SEQ ID NO:20) CACCACGGCCAGTAACAGCGTATACAGTGAT FN5F CAGCGAGCTCCAAGCCAATCTCGA (SEQ ID NO:21) TTAACTACCGT FN5RCGGGATCCTCGAGTTACTAGGTAC (SEQ ID NO:22) GGTAGTTAATCGA FN5R′CGGGATCCACGCGTGCCACCGGTA (SEQ ID NO:23) CGGTAGTTAATCGA gene3FCGGGATCCACGCGTCCATTCGTTT (SEQ ID NO:24) GTGAATATCAAGGCCAATCG gene3RCCGGAAGCTTTAAGACTCCTTATT (SEQ ID NO:25) ACGCAGTATGTTAGC 38TAABgIIICTGTTACTGGCCGTGAGATCTAAC (SEQ ID NO:26) CAGCGAGCTCCA BC3GATCAGCTGGGATGCTCCTNNKNN (SEQ ID NO:27) KNNKNNKNNKTATTACCGTATCAC GTA FG2TGTATACGCTGTTACTGGCNNKNN (SEQ ID NO:28) KNNKNNKNNKNNKNNKTCCAAGCCAATCTCGAT FG3 CTGTATACGCTGTTACTGGCNNKN (SEQ ID NO:29)NKNNKNNKCCAGCGAGCTCCAAG FG4 CATCACTGTATACGCTGTTACTNN (SEQ ID NO:30)KNNKNNKNNKNNKTCCAAGCCAAT CTC

[0121] Structure and stability measurements

[0122] Structures of Abs were analyzed using quantitative methods (e.g.,DSSP (Kabsch & Sander, 1983) and PDBfit (D. McRee, The Scripps ResearchInstitute)) as well as computer graphics (e.g., Quanta (MolecularSimulations) and What if (G. Vriend, European Molecular BiologyLaboratory)) to superimpose the strand-loop-strand structures of Abs andFn3.

[0123] The stability of monobodies was determined by measuringtemperature- and chemical denaturant-induced unfolding reactions (Paceet al., 1989). The temperature-induced unfolding reaction was measuredusing a circular dichroism (CD) polarimeter. Ellipticity at 222 and 215nm was recorded as the sample temperature was slowly raised. Sampleconcentrations between 10 and 50 μM were used. After the unfoldingbaseline was established, the temperature was lowered to examine thereversibility of the unfolding reaction. Free energy of unfolding wasdetermined by fitting data to the equation for the two-state transition(Becktel & Schellman, 1987; Pace et al., 1989). Nonlinear least-squaresfitting was performed using the program Igor (WaveMetrics) on aMacintosh computer.

[0124] The structure and stability of two selected mutant Fn3s werestudied; the first mutant was D1.3-4 (Table 2) and the second was amutant called AS40 which contains four mutations in the BC loop(A²⁶V²⁷T²⁸V²⁹)→TQRQ). AS40 was randomly chosen from the BC loop librarydescribed above. Both mutants were expressed as soluble proteins in E.coli and were concentrated at least to 1 mM, permitting NMR studies.

[0125] The mid-point of the thermal denaturation for both mutants wasapproximately 69° C., as compared to approximately 79° C. for thewild-type protein. The results indicated that the extensive mutations atthe two surface loops did not drastically decrease the stability of Fn3,and thus demonstrated the feasibility of introducing a large number ofmutations in both loops.

[0126] Stability was also determined by guanidinium chloride (GdnCl)-and urea-induced unfolding reactions. Preliminary unfolding curves wererecorded using a fluorometer equipped with a motor-driven syringe; GdnClor urea were added continuously to the protein solution in the cuvette.Based on the preliminary unfolding curves, separate samples containingvarying concentration of a denaturant were prepared and fluorescence(excitation at 290 nm, emission at 300-400 nm) or CD (ellipticity at 222and 215 nm) were measured after the samples were equilibrated at themeasurement temperature for at least one hour. The curve was fitted bythe least-squares method to the equation for the two-state model(Santoro & Bolen, 1988; Koide et al., 1993). The change in proteinconcentration was compensated if required.

[0127] Once the reversibility of the thermal unfolding reaction isestablished, the unfolding reaction is measured by a Microcal MC-2differential scanning calorimeter (DSC). The cell (˜1.3 ml) will befilled with FnAb solution (0.1-1 mM) and ΔCp (=ΔH/ΔT) will be recordedas the temperature is slowly raised. T_(m) (the midpoint of unfolding),ΔH of unfolding and ΔG of unfolding is determined by fitting thetransition curve (Privalov & Potekhin, 1986) with the Origin softwareprovided by Microcal.

[0128] Thermal unfolding

[0129] A temperature-induced unfolding experiment on Fn3 was performedusing circular dichroism (CD) spectroscopy to monitor changes insecondary structure. The CD spectrum of the native Fn3 shows a weaksignal near 222 nm (FIG. 3A), consistent with the predominantlyβ-structure of Fn3 (Perczel et al., 1992). A cooperative unfoldingtransition is observed at 80-90° C., clearly indicating high stabilityof Fn3 (FIG. 3B). The free energy of unfolding could not be determineddue to the lack of a post-transition baseline. The result is consistentwith the high stability of the first Fn3 domain of human fibronectin(Litvinovich et al., 1992), thus indicating that Fn3 domains are ingeneral highly stable.

[0130] Binding assays

[0131] The binding reactions of monobodies were characterizedquantitatively using an isothermal titration calorimeter (ITC) andfluorescence spectroscopy.

[0132] The enthalpy change (ΔH) of binding were measured using aMicrocal Omega ITC (Wiseman et al., 1989). The sample cell (˜1.3 ml) wasfilled with Monobody solution (<100 μM, changed according to K_(d)), andthe reference cell filled with distilled water; the system wasequilibrated at a given temperature until a stable baseline is obtained;5-20 μl of ligand solution (≦2 mM) was injected by a motor-drivensyringe within a short duration (20 sec) followed by an equilibrationdelay (4 minutes); the injection was repeated and heatgeneration/absorption for each injection was measured. From the changein the observed heat change as a function of ligand concentration, ΔHand K_(d) was determined (Wiseman et al., 1989). ΔG and ΔS of thebinding reaction was deduced from the two directly measured parameters.Deviation from the theoretical curve was examined to assess nonspecific(multiple-site) binding. Experiments were also be performed by placing aligand in the cell and titrating with an FnAb. It should be emphasizedthat only ITC gives direct measurement of ΔH, thereby making it possibleto evaluate enthalpic and entropic contributions to the binding energy.ITC was successfully used to monitor the binding reaction of the D1.3 Ab(Tello et al., 1993; Bhat et al., 1994).

[0133] Intrinsic fluorescence is monitored to measure binding reactionswith K_(d) in the sub-μM range where the determination of K_(d) by ITCis difficult. Trp fluorescence (excitation at˜290 nm, emission at300-350 nm) and Tyr fluorescence (excitation at˜260 nm, emission at˜303nm) is monitored as the Fn3-mutant solution (≦10 μM) is titrated withligand solution (≦100 μM). K_(d) of the reaction is determined by thenonlinear least-squares fitting of the bimolecular binding equation.Presence of secondary binding sites is examined using Scatchardanalysis. In all binding assays, control experiments are performedbusing wild-type Fn3 (or unrelated monobodies) in place of monobodies ofinterest.

[0134] II. Production of Fn3 mutants with high affinity and specificityMonobodies

[0135] Library screening was carried out in order to select monobodiesthat bind to specific ligands. This is complementary to the modelingapproach described above. The advantage of combinatorial screening isthat one can easily produce and screen a large number of variants(≧10⁸), which is not feasible with specific mutagenesis (“rationaldesign”) approaches. The phage display technique (Smith, 1985; O'Neil &Hoess, 1995) was used to effect the screening processes. Fn3 was fusedto a phage coat protein (pIII) and displayed on the surface offilamentous phages. These phages harbor a single-stranded DNA genomethat contains the gene coding the Fn3 fusion protein. The amino acidsequence of defined regions of Fn3 were randomized using a degeneratenucleotide sequence, thereby constructing a library. Phages displayingFn3 mutants with desired binding capabilities were selected in vitro,recovered and amplified. The amino acid sequence of a selected clone canbe identified readily by sequencing the Fn3 gene of the selected phage.The protocols of Smith (Smith & Scott, 1993) were followed with minormodifications.

[0136] The objective was to produce Monobodies which have high affinityto small protein ligands. HEL and the B1 domain of staphylococcalprotein G (hereafter referred to as protein G) were used as ligands.Protein G is small (56 amino acids) and highly stable (Minor & Kim,1994; Smith et al., 1994). Its structure was determined by NMRspectroscopy (Gronenborn et al., 1991) to be a helix packed against afour-strand β-sheet. The resulting FnAb-protein G complexes (˜150residues) is one of the smallest protein-protein complexes produced todate, well within the range of direct NMR methods. The small size, thehigh stability and solubility of both components and the ability tolabel each with stable isotopes (¹³C and ¹⁵N; see below for protein G)make the complexes an ideal model system for NMR studies onprotein-protein interactions.

[0137] The successful loop replacement of Fn3 (the mutant D1.3-4)demonstrate that at least ten residues can be mutated without the lossof the global fold. Based on this, a library was first constructed inwhich only residues in the FG loop are randomized. After results of loopreplacement experiments on the BC loop were obtained, mutation siteswere extended that include the BC loop and other sites.

[0138] Construction of Fn3 phage display system

[0139] An M13 phage-based expression vector pASM1 has been constructedas follows: an oligonucleotide coding the signal peptide of OmpT wascloned at the 5′ end of the Fn3 gene; a gene fragment coding theC-terminal domain of M13 pIII was prepared from the wild-type gene IIIgene of M13 mp18 using PCR (Corey et al., 1993) and the fragment wasinserted at the 3′ end of the OmpT-Fn3 gene; a spacer sequence has beeninserted between Fn3 and pIII. The resultant fragment (OmpT-Fn3-pIII)was cloned in the multiple cloning site of M13 mp18, where the fusiongene is under the control of the lac promoter. This system will producethe Fn3-pIII fusion protein as well as the wild-type pIII protein. Theco-expression of wild-type pIII is expected to reduce the number offusion pIII protein, thereby increasing the phage infectivity (Corey etal., 1993) (five copies of pIII are present on a phage particle). Inaddition, a smaller number of fusion pIII protein may be advantageous inselecting tight binding proteins, because the chelating effect due tomultiple binding sites should be smaller than that with all five copiesof fusion pIII (Bass et al., 1990). This system has successfullydisplayed the serine protease trypsin (Corey et al., 1993). Phages wereproduced and purified using E. coli K91kan (Smith & Scott, 1993)according to a standard method (Sambrook et al., 1989) except that phageparticles were purified by a second polyethylene glycol precipitationand acid precipitation.

[0140] Successful display of Fn3 on fusion phages has been confirmed byELISA using an Ab against fibronectin (Sigma), clearly indicating thatit is feasible to construct libraries using this system.

[0141] An alternative system using the fUSE5 (Parmley & Smith, 1988) mayalso be used. The Fn3 gene is inserted to fUSE5 using the SfiIrestriction sites introduced at the 5′- and 3′- ends of the Fn3 genePCR. This system displays only the fusion pIII protein (up to fivecopies) on the surface of a phage. Phages are produced and purified asdescribed (Smith & Scott, 1993). This system has been used to displaymany proteins and is robust. The advantage of fUSE5 is its low toxicity.This is due to the low copy number of the replication form (RF) in thehost, which in turn makes it difficult to prepare a sufficient amount ofRF for library construction (Smith & Scott, 1993).

[0142] Construction of libraries

[0143] The first library was constructed of the Fn3 domain displayed onthe surface of M13 phage in which seven residues (77-83) in the FG loop(FIG. 4D) were randomized. Randomization will be achieved by the use ofan oligonucleotide containing degenerated nucleotide sequence. Adouble-stranded nucleotide was prepared by the same protocol as for genesynthesis (see above) except that one strand had an (NNK)₆(NNG) sequenceat the mutation sites, where N corresponds to an equimolar mixture of A,T, G and C and K corresponds to an equimolar mixture of G and T. The(NNG) codon at residue 83 was required to conserve the SacI restrictionsite (FIG. 2). The (NNK) codon codes all of the 20 amino acids, whilethe NNG codon codes 14. Therefore, this library contained˜10⁹independent sequences. The library was constructed by ligating thedouble-stranded nucleotide into the wild-type phage vector, pASM1, andthe transfecting E. coli XL1 blue (Stratagene) using electroporation.XL1 blue has the lacIq phenotype and thus suppresses the expression ofthe Fn3-pIII fusion protein in the absence of lac inducers. The initiallibrary was propagated in this way, to avoid selection against toxicFn3-pIII clones. Phages displaying the randomized Fn3-III fusion proteinwere prepared by propagating phages with K91kan as the host. K91kan doesnot suppress the production of the fusion protein, because it does nothave lacI^(q). Another library was also generated in which the BC loop(residues 26-20) was randomized.

[0144] Selection of displayed Monobodies

[0145] Screening of Fn3 phage libraries was performed using thebiopanning protocol (Smith & Scott, 1993); a ligand is biotinylated andthe strong biotin-streptavidin interaction was used to immobilize theligand on a streptavidin-coated dish. Experiments were performed at roomtemperature (˜22° C.). For the initial recovery of phages from alibrary, 10 μg of a biotinylated ligand were immobilized on astreptavidin-coated polystyrene dish (35 mm, Falcon 1008) and then aphage solution (containing˜10¹¹ pfu (plaque-forming unit)) was added.After washing the dish with an appropriate buffer (typically TBST,Tris-HCl (50 mM, pH 7.5), NaCl (150 mM) and Tween 20 (0.5%)), boundphages were eluted by one or combinations of the following conditions:low pH, an addition of a free ligand, urea (up to 6 M) and, in the caseof anti-protein G Monobodies, cleaving the protein G-biotin linker bythrombin. Recovered phages were amplified using the standard protocolusing K91kan as the host (Sambrook et al., 1989). The selection processwere repeated 3-5 times to concentrate positive clones. From the secondround on, the amount of the ligand were gradually decreased (to ˜1 μg)and the biotinylated ligand were mixed with a phage solution beforetransferring a dish (G. P. Smith, personal communication). After thefinal round, 10-20 clones were picked, and their DNA sequence will bedetermined. The ligand affinity of the clones were measured first by thephage-ELISA method (see below).

[0146] To suppress potential binding of the Fn3 framework (backgroundbinding) to a ligand, wild-type Fn3 may be added as a competitor in thebuffers. In addition, unrelated proteins (e.g., bovine serum albumin,cytochrome c and RNase A) may be used as competitors to select highlyspecific Monobodies.

[0147] Binding assay

[0148] The binding affinity of Monobodies on phage surface ischaracterized semi-quantitatively using the phage ELISA technique (Li etal., 1995). Wells of microtiter plates (Nunc) are coated with a ligandprotein (or with streptavidin followed by the binding of a biotinylatedligand) and blocked with the Blotto solution (Pierce). Purified phages(˜10¹⁰ pfu) originating from single plaques (M13)/colonies (fUSE5) areadded to each well and incubated overnight at 4° C. After washing wellswith an appropriate buffer (see above), bound phages are detected by thestandard ELISA protocol using anti-M13 Ab (rabbit, Sigma) andanti-rabbit Ig-peroxidase conjugate (Pierce) or using anti-M13Ab-peroxidase conjugate (Pharmacia). Colormetric assays are performedusing TMB (3,3′,5,5′-tetramethylbenzidine, Pierce). The high affinity ofprotein G to immunoglobulins present a special problem; Abs cannot beused in detection. Therefore, to detect anti-protein G Monobodies,fusion phages are immobilized in wells and the binding is then measuredusing biotinylated protein G followed by the detection usingstreptavidin-peroxidase conjugate.

[0149] Production of soluble Monobodies

[0150] After preliminary characterization of mutant Fn3s using phageELISA, mutant genes are subcloned into the expression vector pEW1.Mutant proteins are produced as His.tag fusion proteins and purified,and their conformation, stability and ligand affinity are characterized.

[0151] III. Increased Stability of Fn3 Scaffolds

[0152] The definition of “higher stability” of a protein is the abilityof a protein to retain its three-dimensional structure required forfunction at a higher temperature (in the case of thermal denaturation),and in the presence of a higher concentration of a denaturing chemicalreagent such as guanidine hydrochloride. This type of “stability” isgenerally called “conformational stability.” It has been shown thatconformational stability is correlated with resistance againstproteolytic degradation, i.e., breakdown of protein in the body(Kamtekar et al. 1993).

[0153] Improving the conformational stability is a major goal in proteinengineering. Here, mutations have been developed by the inventor thatenhance the stability of the fibronectin type III domain (Fn3). Theinventor has developed a technology in which Fn3 is used as a scaffoldto engineer artificial binding proteins (Koide et al., 1998). It hasbeen shown that many residues in the surface loop regions of Fn3 can bemutated without disrupting the overall structure of the Fn3 molecule,and that variants of Fn3 with a novel binding function can be engineeredusing combinatorial library screening (Koide et al., 1998). The inventorfound that, although Fn3 is an excellent scaffold, Fn3 variants thatcontain large number of mutations are destablized against chemicaldenaturation, compared to the wild-type Fn3 protein (Koide et al.,1998). Thus, as the number of mutated positions are mutated in order toengineer a new binding function, the stability of such Fn3 variantsfurther decreases, ultimately leading to marginally stable proteins.Because artificial binding proteins must maintain theirthree-dimensional structure to be functional, stability limits thenumber of mutations that can be introduced in the scaffold. Thus,modifications of the Fn3 scaffold that increase its stability are usefulin that they allow one to introduce more mutations for better function,and that they make it possible to use Fn3-based engineered proteins in awider range of applications.

[0154] The inventor found that wild-type Fn3 is more stable at acidic pHthan at neutral pH (Koide et al., 1998). The pH dependence of Fn3stability is characterized in FIG. 18. The pH dependence curve has anapparent transition midpoint near pH 4 (FIG. 18). These results suggestthat by identifying and removing destablizing interactions in Fn3 one isable to improve the stability of Fn3 at neutral pH. It should be notedthat most applications of engineered Fn3, such as diagnostics,therapeutics and catalysts, are expected to be used near neutral pH, andthus it is important to improve the stability at neutral pH. Studies byother investigators have demonstrated that the optimization of surfaceelectrostatic properties can lead to a substantial increase in proteinstability (Perl et al. 2000, Spector et al. 1999, Loladze et al. 1999,Grimsley et al. 1999).

[0155] The pH dependence of Fn3 stability suggests that amino acids withpK_(a) near 4 are involved in the observed transition. The carboxylgroups of aspartic acid (Asp) and glutamic acid (Glu) have pK_(a) inthis range (Creighton, T. E. 1993). It is well known that if a carboxylgroup has unfavorable (i.e. destabilizing) interactions in a protein,its pK_(a) is shifted to a higher value from its standard, unperturbedvalue (Yang and Honig 1992). Thus, the pK_(a) values of all carboxylgroups in Fn3 were determined using nuclear magnetic resonance (NMR)spectrosocpy, to identify carboxyl groups with unusual pK_(a)'s, asshown below.

[0156] First, the ¹³C resonance for the carboxyl carbon of each Asp andGlu residue were assigned (FIG. 19). Next pH titration of 13C resonanceswas performed for these groups (FIG. 20). The pK_(a) values for theseresidues are listed in Table 3. TABLE 3 pK_(a) values for Asp and Gluresidues in Fn3. Residue pK_(a) E9 5.09 E38 3.79 E47 3.94 D3 3.66 D73.54, 5.54* D23 3.54, 5.25* D67 4.18 D80 3.40

[0157] These results show that Asp 7 and 23, and Glu 9 have up-shiftedpK_(a)'s with respect to their unperturbed pK_(a)'s (approximately 4.0),indicating that these residues are involved in unfavorable interactions.In contrast, the other Asp and Glu residues have pK_(a)'s close to therespective unperturbed values, indicating that the carboxyl groups ofthese residues do not significantly contribute to the stability of Fn3.

[0158] In the three-dimensional structure of Fn3 (Main et al. 1992), Asp7 and 23, and Glu 9 form a patch on the surface (FIG. 21), with Asp 7centrally located in the patch. This spatial proximity of thesenegatively charged residues explains why these residues have unfavorableinteractions in Fn3. At low pH where these residues are protonated andneutral, the unfavorable interactions are expected to be mostlyrelieved. At the same time, the structure suggests that the stability ofFn3 at neutral pH could be improved if the electrostatic repulsionbetween these three residues is removed. Because Asp 7 is centrallylocated among the three residues, it was decided to mutate Asp 7. Twomutants were prepared, D7N and D7K (i.e., the aspartic acid at aminoacid residue number 7 was substituted with an asparagine residue or alysine residue, respectively). The former replaces the negative chargewith a neutral residue of virtually the same size. The latter places apositive charge at residue 7.

[0159] The degrees of stability of the mutant proteins werecharacterized in thermal and chemical denaturation measurements. Inthermal denaturation measurements, denaturation of the Fn3 proteins wasmonitored using circular dichroism spectroscopy at the wavelength of 227nm. All the proteins underwent a cooperative transition (FIG. 22). Fromthe transition curves, the midpoints of the transition (T_(m)) for thewild-type, D7N and D7K were determined to be 62, 69 and 70 ° C. in 0.02M sodium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride and6.2 M urea. Thus, the mutations increased the T_(m) of wild-type Fn3 by7-8° C.

[0160] Chemical denaturation of Fn3 proteins was monitored usingfluorescence emission from the single Trp residue of Fn3 (FIG. 23). Thefree energies of unfolding in the absence of guanidine HCl (ΔG⁰) weredetermined to be 7.4, 8.1 and 8.0 kcal/mol for the wild-type, D7N andD7K, respectively (a larger ΔG⁰ indicates a higher stability). The twomutants were again found to be more stable than the wild-type protein.

[0161] These results show that a point mutation on the surface cansignificantly enhance the stability of Fn3. Because these mutations areon the surface, they minimally alter the structure of Fn3, and they canbe easily introduced to other, engineered Fn3 proteins. In addition,mutations at Glu 9 and/or Asp 23 also enhance the stability of Fn3.Furthermore, mutations at one or more of these three residues can becombined.

[0162] Thus, Fn3 is the fourth example of a monomericimmunoglobulin-like scaffold that can be used for engineering bindingproteins. Successful selection of novel binding proteins have also beenbased on minibody, tendamistat and “camelized” immunoglobulin VH domainscaffolds (Martin et al., 1994; Davies & Riechmann, 1995; McConnell &Hoess, 1995). The Fn3 scaffold has advantages over these systems.Bianchi et al. reported that the stability of a minibody was 2.5kcal/mol, significantly lower than that of Ubi4-K. No detailedstructural characterization of minibodies has been reported to date.Tendamistat and the VH domain contain disulfide bonds, and thuspreparation of correctly folded proteins may be difficult. Davies andRiechmann reported that the yields of their camelized VH domains wereless than 1 mg per liter culture (Davies & Riechmann, 1996).

[0163] Thus, the Fn3 framework can be used as a scaffold for molecularrecognition. Its small size, stability and well-characterized structuremake Fn3 an attractive system. In light of the ubiquitous presence ofFn3 in a wide variety of natural proteins involved in ligand binding,one can engineer Fn3-based binding proteins to different classes oftargets.

[0164] The following examples are intended to illustrate but not limitthe invention.

EXAMPLE I Construction of the Fn3 Gene

[0165] A synthetic gene for tenth Fn3 of fibronectin (FIG. 1) wasdesigned on the basis of amino acid residue 1416-1509 of humanfibronectin (Komblihtt, et al., 1985) and its three dimensionalstructure (Main, et al., 1992). The gene was engineered to includeconvenient restriction sites for mutagenesis and the so-called“preferred codons” for high level protein expression (Gribskov, et al.,1984) were used. In addition, a glutamine residue was inserted after theN-terminal methionine in order to avoid partial processing of theN-terminal methionine which often degrades NMR spectra (Smith, et al.,1994). Chemical reagents were of the analytical grade or better andpurchased from Sigma Chemical Company and J. T. Baker, unless otherwisenoted. Recombinant DNA procedures were performed as described in“Molecular Cloning” (Sambrook, et al., 1989), unless otherwise stated.Custom oligonucleotides were purchased from Operon Technologies.Restriction and modification enzymes were from New England Biolabs.

[0166] The gene was assembled in the following manner. First, the genesequence (FIG. 5) was divided into five parts with boundaries atdesigned restriction sites: fragment 1, NdeI-PstI (oligonucleotides FN1Fand FN1R (Table 2); fragment 2, PstI-EcoRI (FN2F and FN2R); fragment 3,EcoRI-SalI (FN3F and FN3R); fragment 4, SalI-SacI (FN4F and FN4R);fragment 5, SacI-BamHI (FN5F and FN5R). Second, for each part, a pair ofoligonucleotides which code opposite strands and have complementaryoverlaps of approximately 15 bases was synthesized. Theseoligonucleotides were designated FN1F-FN5R and are shown in Table 2.Third, each pair (e.g., FN1F and FN1R) was annealed and single-strandregions were filled in using the Klenow fragment of DNA polymerase.Fourth, the double stranded oligonucleotide was digested with therelevant restriction enzymes at the termini of the fragment and clonedinto the pBlueScript SK plasmid (Stratagene) which had been digestedwith the same enzymes as those used for the fragments. The DNA sequenceof the inserted fragment was confirmed by DNA sequencing using anApplied Biosystems DNA sequencer and the dideoxy termination protocolprovided by the manufacturer. Last, steps 2-4 were repeated to obtainthe entire gene.

[0167] The gene was also cloned into the pET3a and pET15b (Novagen)vectors (pAS45 and pAS25, respectively). The maps of the plasmids areshown in FIGS. 6 and 7. E. coli BL21 (DE3) (Novagen) containing thesevectors expressed the Fn3 gene under the control of bacteriophage T7promotor (Studier, et al., 1990); pAS24 expresses the 96-residue Fn3protein only, while pAS45 expresses Fn3 as a fusion protein withpoly-histidine peptide (His.tag). High level expression of the Fn3protein and its derivatives in E. coli was detected as an intense bandon SDS-PAGE stained with CBB.

[0168] The binding reaction of the monobodies is characterizedquantitatively by means of fluorescence spectroscopy using purifiedsoluble monobodies.

[0169] Intrinsic fluorescence is monitored to measure binding reactions.Trp fluorescence (excitation at˜290 nm, emission at 300 350 nm) and Tyrfluorescence (excitation at˜260 nm, emission at˜303 nm) is monitored asthe Fn3-mutant solution (≦100 μM) is titrated with a ligand solution.When a ligand is fluorescent (e.g. fluorescein), fluorescence from theligand may be used. K_(d) of the reaction will be determined by thenonlinear least-squares fitting of the bimolecular binding equation.

[0170] If intrinsic fluorescence cannot be used to monitor the bindingreaction, monobodies are labeled with fluorescein-NHS (Pierce) andfluorescence polarization is used to monitor the binding reaction (Burkeet al., 1996).

EXAMPLE II Modifications to Include Restriction Sites in the Fn3 Gene

[0171] The restriction sites were incorporated in the synthetic Fn3 genewithout changing the amino acid sequence Fn3. The positions of therestriction sites were chosen so that the gene construction could becompleted without synthesizing long (>60 bases) oligonucleotides and sothat two loop regions could be mutated (including by randomization) bythe cassette mutagenesis method (i.e., swapping a fragment with anothersynthetic fragment containing mutations). In addition, the restrictionsites were chosen so that most sites were unique in the vector for phagedisplay. Unique restriction sites allow one to recombine monobody cloneswhich have been already selected in order to supply a larger sequencespace.

EXAMPLE III Construction of M13 Phage Display Libraries

[0172] A vector for phage display, pAS38 (for its map, see FIG. 8) wasconstructed as follows. The XbaI-BamHI fragment of pET12a encoding thesignal peptide of OmpT was cloned at the 5′ end of the Fn3 gene. TheC-terminal region (from the FN5F and FN5R oligonucleotides, see Table 2)of the Fn3 gene was replaced with a new fragment consisting of the FN5Fand FN5R′ oligonucleotides (Table 2) which introduced a MluI site and alinker sequence for making a fusion protein with the pIII protein ofbacteriophage M13. A gene fragment coding the C-terminal domain of M13pIII was prepared from the wild-type gene III of M13mp18 using PCR(Corey, et al., 1993) and the fragment was inserted at the 3′ end of theOmpT-Fn3 fusion gene using the MluI and HindIII sites.

[0173] Phages were produced and purified using a helper phage, M13K07,according to a standard method (Sambrook, et al., 1989) except thatphage particles were purified by a second polyethylene glycolprecipitation. Successful display of Fn3 on fusion phages was confirmedby ELISA (Harlow & Lane, 1988) using an antibody against fibronectin(Sigma) and a custom anti-FN3 antibody (Cocalico Biologicals, PA, USA).

EXAMPLE IV Libraries Containing Loop Variegations in the AB Loop

[0174] A nucleic acid phage display library having variegation in the ABloop is prepared by the following methods. Randomization is achieved bythe use of oligonucleotides containing degenerated nucleotide sequence.Residues to be variegated are identified by examining the X-ray and NMRstructures of Fn3 (Protein Data Bank accession numbers, 1FNA and 1TTF,respectively). Oligonucleotides containing NNK (N and K here denote anequimolar mixture of A, T, G, and C and an equimolar mixture of G and T,respectively) for the variegated residues are synthesized (seeoligonucleotides BC3, FG2, FG3, and FG4 in Table 2 for example). The NNKmixture codes for all twenty amino acids and one termination codon(TAG). TAG, however, is suppressed in the E. coli XL-1 blue.Single-stranded DNAs of pAS38 (and its derivatives) are prepared using astandard protocol (Sambrook, et al., 1989).

[0175] Site-directed mutagenesis is performed following publishedmethods (see for example, Kunkel, 1985) using a Muta-Gene kit (BioRad).The libraries are constructed by electroporation of E. coli XL- 1 Blueelectroporation competent cells (200 μl; Stratagene) with 1 μg of theplasmid DNA using a BTX electrocell manipulator ECM 395 1 mm gapcuvette. A portion of the transformed cells is plated on an LB-agarplate containing ampicillin (100 μg/ml) to determine the transformationefficiency. Typically, 3×10⁸ transformants are obtained with 1 μg ofDNA, and thus a library contains 10⁸ to 10⁹ independent clones. Phagemidparticles were prepared as described above.

EXAMPLE V Loop Variegations in the BC, CD, DE, EF or FG Loop

[0176] A nucleic acid phage display library having five variegatedresidues (residues number 26-30) in the BC loop, and one having sevenvariegated residues (residue numbers 78-84) in the FG loop, was preparedusing the methods described in Example IV above. Other nucleic acidphage display libraries having variegation in the CD, DE or EF loop canbe prepared by similar methods.

EXAMPLE VI Loop Variegations in the FG and BC Loop

[0177] A nucleic acid phage display library having seven variegatedresidues (residues number 78-84) in the FG loop and five variegatedresidues (residue number 26-30) in the BC loop was prepared.Variegations in the BC loop were prepared by site-directed mutagenesis(Kunkel, et al.) using the BC3 oligonucleotide described in Table 1.Variegations in the FG loop were introduced using site-directedmutagenesis using the BC loop library as the starting material, therebyresulting in libraries containing variegations in both BC and FG loops.The oligonucleotide FG2 has variegating residues 78-84 andoligonucleotide FG4 has variegating residues 77-81 and a deletion ofresidues 82-84.

[0178] A nucleic acid phage display library having five variegatedresidues (residues 78-84) in the FG loop and a three residue deletion(residues 82-84) in the FG loop, and five variegated residues (residues26-30) in the BC loop, was prepared. The shorter FG loop was made in anattempt to reduce the flexibility of the FG loop; the loop was shown tobe highly flexible in Fn3 by the NMR studies of Main, et al. (1992). Ahighly flexible loop may be disadvantageous to forming a binding sitewith a high affinity (a large entropy loss is expected upon the ligandbinding, because the flexible loop should become more rigid). Inaddition, other Fn3 domains (besides human) have shorter FG loops (forsequence alignment, see FIG. 12 in Dickinson, et al. (1994)).

[0179] Randomization was achieved by the use of oligonucleotidescontaining degenerate nucleotide sequence (oligonucleotide BC3 forvariegating the BC loop and oligonucleotides FG2 and FG4 for variegatingthe FG loops).

[0180] Site-directed mutagenesis was performed following publishedmethods (see for example, Kunkel, 1985). The libraries were constructedby electrotransforming E. coli XL-1 Blue (Stratagene). Typically alibrary contains 10⁸ to 10⁹ independent clones. Library 2 contains fivevariegated residues in the BC loop and seven variegated residues in theFG loop. Library 4 contains five variegated residues in each of the BCand FG loops, and the length of the FG loop was shortened by threeresidues.

EXAMPLE VII Fd Phage Display Libraries Constructed with LoopVariegations

[0181] Phage display libraries are constructed using the fd phage as thegenetic vector. The Fn3 gene is inserted in fUSE5 (Parmley & Smith,1988) using SfiI restriction sites which are introduced at the 5′ and 3′ends of the Fn3 gene using PCR. The expression of this phage results inthe display of the fusion pIII protein on the surface of the fd phage.Variegations in the Fn3 loops are introduced using site-directedmutagenesis as described hereinabove, or by subcloning the Fn3 librariesconstructed in M13 phage into the fUSE5 vector.

EXAMPLE VIII Other Phage Display Libraries

[0182] T7 phage libraries (Novagen, Madison, Wis.) and bacterial piliexpression systems (Invitrogen) are also useful to express the Fn3 gene.

EXAMPLE IX Isolation of Polypeptides Which Bind to MacromolecularStructures

[0183] The selection of phage-displayed monobodies was performedfollowing the protocols of Barbas and coworkers (Rosenblum & Barbas,1995). Briefly, approximately 1 μg of a target molecule (“antigen”) insodium carbonate buffer (100 mM, pH 8.5) was immobilized in the wells ofa microtiter plate (Maxisorp, Nunc) by incubating overnight at 4° C. inan air tight container. After the removal of this solution, the wellswere then blocked with a 3% solution of BSA (Sigma, Fraction V) in TBSby incubating the plate at 37° C. for 1 hour. A phagemid librarysolution (50 μl) containing approximately 10¹² colony forming units(cfu) of phagemid was absorbed in each well at 37° C. for 1 hour. Thewells were then washed with an appropriate buffer (typically TBST, 50 mMTris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Tween20) three times (once forthe first round). Bound phage were eluted by an acidic solution(typically, 0.1 M glycine-HCl, pH 2.2; 50 μl) and recovered phage wereimmediately neutralized with 3 μl of Tris solution. Alternatively, boundphage were eluted by incubating the wells with 50 μl of TBS containingthe antigen (1-10 μM). Recovered phage were amplified using the standardprotocol employing the XL1 Blue cells as the host (Sambrook, et al.).The selection process was repeated 5-6 times to concentrate positiveclones. After the final round, individual clones were picked and theirbinding affinities and DNA sequences were determined.

[0184] The binding affinities of monobodies on the phage surface werecharacterized using the phage ELISA technique (Li, et al., 1995). Wellsof microtiter plates (Nunc) were coated with an antigen and blocked withBSA. Purified phages (10⁸-10¹¹ cfu) originating from a single colonywere added to each well and incubated 2 hours at 37° C. After washingwells with an appropriate buffer (see above), bound phage were detectedby the standard ELISA protocol using anti-M13 antibody (rabbit, Sigma)and anti-rabbit Ig-peroxidase conjugate (Pierce). Colorimetric assayswere performed using Turbo-TMB (3,3′,5,5′-tetramethylbenzidine, Pierce)as a substrate.

[0185] The binding affinities of monobodies on the phage surface werefurther characterized using the competition ELISA method(Djavadi-Ohaniance, et al., 1996). In this experiment, phage ELISA isperformed in the same manner as described above, except that the phagesolution contains a ligand at varied concentrations. The phage solutionwas incubated a 4° C. for one hour prior to the binding of animmobilized ligand in a microtiter plate well. The affinities of phagedisplayed monobodies are estimated by the decrease in ELISA signal asthe free ligand concentration is increased.

[0186] After preliminary characterization of monobodies displayed on thesurface of phage using phage ELISA, genes for positive clones weresubcloned into the expression vector pAS45. E. coli BL21 (DE3) (Novagen)was transformed with an expression vector (pAS45 and its derivatives).Cells were grown in M9 minimal medium and M9 medium supplemented withBactotryptone (Difco) containing ampicillin (200 μg/ml). For isotopiclabeling, ¹⁵N NH₄Cl and/or ¹³C glucose replaced unlabeled components.Stable isotopes were purchased from Isotec and Cambridge Isotope Labs.500 ml medium in a 2 l baffle flask was inoculated with 10 ml ofovernight culture and agitated at approximately 140 rpm at 37° C. IPTGwas added at a final concentration of 1 mM to induce protein expressionwhen OD(600 nm) reached approximately 1.0. The cells were harvested bycentrifugation 3 hours after the addition of IPTG and kept frozen at−70° C. until used.

[0187] Fn3 and monobodies with His.tag were purified as follows. Cellswere suspended in 5 ml/(g cell) of 50 mM Tris (pH 7.6) containing 1 mMphenylmethylsulfonyl fluoride. HEL (Sigma, 3× crystallized) was added toa final concentration of 0.5 mg/ml. After incubating the solution for 30min at 37° C., it was sonicated so as to cause cell breakage three timesfor 30 seconds on ice. Cell debris was removed by centrifugation at15,000 rpm in an Sorval RC-2B centrifuge using an SS-34 rotor.Concentrated sodium chloride is added to the solution to a finalconcentration of 0.5 M. The solution was then applied to a 1 ml HisTrap™chelating column (Pharmacia) preloaded with nickel chloride (0.1 M, 1ml) and equilibrated in the Tris buffer (50 mM, pH 8.0) containing 0.5 Msodium chloride. After washing the column with the buffer, the boundprotein was eluted with a Tris buffer (50 mM, pH 8.0) containing 0.5 Mimidazole. The His.tag portion was cleaved off, when required, bytreating the fusion protein with thrombin using the protocol supplied byNovagen (Madison, Wis.). Fn3 was separated from the His.tag peptide andthrombin by a Resources®column (Pharmacia) using a linear gradient ofsodium chloride (0-0.5 M) in sodium acetate buffer (20 mM, pH 5.0).

[0188] Small amounts of soluble monobodies were prepared as follows.XL-1 Blue cells containing pAS38 derivatives (plasmids coding Fn3-pIIIfusion proteins) were grown in LB media at 37° C. with vigorous shakinguntil OD(600 nm) reached approximately 1.0; IPTG was added to theculture to a final concentration of 1 mM, and the cells were furthergrown overnight at 37° C. Cells were removed from the medium bycentrifugation, and the supernatant was applied to a microtiter wellcoated with a ligand. Although XL-1 Blue cells containing pAS38 and itsderivatives express FN3-pIII fusion proteins, soluble proteins are alsoproduced due to the cleavage of the linker between the Fn3 and pIIIregions by proteolytic activities of E. coli (Rosenblum & Barbas, 1995).Binding of a monobody to the ligand was examined by the standard ELISAprotocol using a custom antibody against Fn3 (purchased from CocalicoBiologicals, Reamstown, Pa.). Soluble monobodies obtained from theperiplasmic fraction of E. coli cells using a standard osmotic shockmethod were also used.

EXAMPLE X Ubiquitin Binding Monobody

[0189] Ubiquitin is a small (76 residue) protein involved in thedegradation pathway in eurkaryotes. It is a single domain globularprotein. Yeast ubiquitin was purchased from Sigma Chemical Company andwas used without further purification.

[0190] Libraries 2 and 4, described in Example VI above, were used toselect ubiquitin-binding monobodies. Ubiquitin (1 μg in 50 μl sodiumbicarbonate buffer (100 mM, pH 8.5)) was immobilized in the wells of amicrotiter plate, followed by blocking with BSA (3% in TBS). Panning wasperformed as described above. In the first two rounds, 1 μg of ubiquitinwas immobilized per well, and bound phage were elute with an acidicsolution. From the third to the sixth rounds, 0.1 μg of ubiquitin wasimmobilized per well and the phage were eluted either with an acidicsolution or with TB S containing 10 μM ubiquitin.

[0191] Binding of selected clones was tested first in the polyclonalmode, i.e., before isolating individual clones. Selected clones from alllibraries showed significant binding to ubiquitin. These results areshown in FIG. 9. The binding to the immobilized ubiquitin of the cloneswas inhibited almost completely by less than 30 μM soluble ubiquitin inthe competition ELISA experiments (see FIG. 10). The sequences of the BCand FG loops of ubiquitin-binding monobodies is shown in Table 4. TABLE4 Sequences of ubiquitin-binding monobodies Occurrence (if more Name BCloop FG loop than one) 211 CARRA RWIPLAK 2 (SEQ ID NO:31) (SEQ ID NO:32)212 CWRRA RWVGLAW (SEQ ID NO:33) (SEQ ID NO:34) 213 CKHRR FADLWWR (SEQID NO:35) (SEQ ID NO:36) 214 CRRGR RGFMWLS (SEQ ID NO:37) (SEQ ID NO:38)215 CNWRR RAYRYRW (SEQ ID NO:39) (SEQ ID NO:40) 411 SRLRR PPWRV 9 (SEQID NO:41) (SEQ ID NO:42) 422 ARWTL RRWWW (SEQ ID NO:43) (SEQ ID NO:44)424 GQRTF RRWWA (SEQ ID NO:45) (SEQ ID NO:46)

EXAMPLE XI Methods for the Immobilization of Small Molecules

[0192] Target molecules were immobilized in wells of a microtiter plate(Maxisorp, Nunc) as described hereinbelow, and the wells were blockedwith BSA. In addition to the use of carrier protein as described below,a conjugate of a target molecule in biotin can be made. The biotinylatedligand can then be immobilized to a microtiter plate well which has beencoated with streptavidin.

[0193] In addition to the use of a carrier protein as described below,one could make a conjugate of a target molecule and biotin (Pierce) andimmobilize a biotinylated ligand to a microtiter plate well which hasbeen coated with streptavidin (Smith and Scott, 1993).

[0194] Small molecules may be conjugated with a carrier protein such asbovine serum albumin (BSA, Sigma), and passively adsorbed to themicrotiter plate well. Alternatively, methods of chemical conjugationcan also be used. In addition, solid supports other than microtiterplates can readily be employed.

EXAMPLE XII Fluorescein Binding Monobody

[0195] Fluorescein has been used as a target for the selection ofantibodies from combinatorial libraries (Barbas, et al. 1992).NHS-fluorescein was obtained from Pierce and used according to themanufacturer's instructions in preparing conjugates with BSA (Sigma).Two types of fluorescein-BSA conjugates were prepared with approximatemolar ratios of 17 (fluorescein) to one (BSA).

[0196] The selection process was repeated 5-6 times to concentratepositive clones. In this experiment, the phage library was incubatedwith a protein mixture (BSA, cytochrome C (Sigma, Horse) and RNaseA(Sigma, Bovine), 1 mg/ml each) at room temperature for 30 minutes, priorto the addition to ligand coated wells. Bound phage were eluted in TBScontaining 10 μM soluble fluorescein, instead of acid elution. After thefinal round, individual clones were picked and their binding affinities(see below) and DNA sequences were determined. TABLE 5 BC FG Clones fromLibrary #2 WT AVTVR (SEQ ID NO:47) RGDSPAS (SEQ ID NO:48) pLB CNWRR (SEQID NO:49) RAYRYRW (SEQ ID NO:50) 24.1 pLB CMWRA (SEQ ID NO:51) RWGMLRR(SEQ ID NO:52) 24.2 pLB ARMRE (SEQ ID NO:53) RWLRGRY (SEQ ID NO:54) 24.3pLB CARRR (SEQ ID NO:55) RRAGWGW (SEQ ID NO:56) 24.4 pLB CNWRR (SEQ IDNO:57) RAYRYRW (SEQ ID NO:58) 24.5 pLB RWRER (SEQ ID NO:59) RHPWTER (SEQID NO:60) 24.6 pLB CNWRR (SEQ ID NO:61) RAYRYRW (SEQ ID NO:62) 24.7 pLBERRVP (SEQ ID NO:63) RLLLWQR (SEQ ID NO:64) 24.8 pLB GRGAG (SEQ IDNO:65) FGSFERR (SEQ ID NO:66) 24.9 pLB CRWTR (SEQ ID NO:67) RRWFDGA (SEQID NO:68) 24.11 pLB CNWRR (SEQ ID NO:69) RAYRYRW (SEQ ID NO:70) 24.12Clones from Library #4 WT AVTVR (SEQ ID NO:71) GRGDS   (SEQ ID NO:72)pLB GQRTF (SEQ ID NO:73) RRWWA   (SEQ ID NO:74) 25.1 pLB GQRTF (SEQ IDNO:75) RRWWA   (SEQ ID NO:76) 25.2 pLB GQRTF (SEQ ID NO:77) RRWWA   (SEQID NO:78) 25.3 pLB LRYRS (SEQ ID NO:79) GWRWR   (SEQ ID NO:80) 25.4 pLBGQRTF (SEQ ID NO:81) RRWWA   (SEQ ID NO:82) 25.5 pLB GQRTF (SEQ IDNO:83) RRWWA   (SEQ ID NO:84) 25.6 pLB LRYRS (SEQ ID NO:85) GWRWR   (SEQID NO:86) 25.7 pLB LRYRS (SEQ ID NO:87) GWRWR   (SEQ ID NO:88) 25.9 pLBGQRTF (SEQ ID NO:89) RRWWA   (SEQ ID NO:90) 25.11 pLB LRYRS (SEQ IDNO:91) GWRWR   (SEQ ID NO:92) 25.12

[0197] Preliminary characterization of the binding affinities ofselected clones were performed using phage ELISA and competition phageELISA (see FIG. 12 (Fluorescein-1) and FIG. 13 (Fluorescein-2)). Thefour clones tested showed specific binding to the ligand-coated wells,and the binding reactions are inhibited by soluble fluorescein (see FIG.13).

EXAMPLE XIII Digoxigenin Binding Monobody

[0198] Digoxigenin-3-O-methyl-carbonyl-e-aminocapronic acid-NHS(Boehringer Mannheim) is used to prepare a digoxigenin-BSA conjugate.The coupling reaction is performed following the manufacturers'instructions. The digoxigenin-BSA conjugate is immobilized in the wellsof a microtiter plate and used for panning. Panning is repeated 5 to 6times to enrich binding clones. Because digoxigenin is sparingly solublein aqueous solution, bound phages are eluted from the well using acidicsolution. See Example XIV.

EXAMPLE XIV TSAC (Transition State Analog Compound) Binding Monobodies

[0199] Carbonate hydrolyzing monobodies are selected as follows. Atransition state analog for carbonate hydrolysis, 4-nitrophenylphosphonate is synthesized by an Arbuzov reaction as describedpreviously (Jacobs and Schultz, 1987). The phosphonate is then coupledto the carrier protein, BSA, using carbodiimide, followed by exhaustivedialysis (Jacobs and Schultz, 1987). The hapten-BSA conjugate isimmobilized in the wells of a microtiter plate and monobody selection isperformed as described above. Catalytic activities of selectedmonobodies are tested using 4-nitrophenyl carbonate as the substrate.

[0200] Other haptens useful to produce catalytic monobodies aresummarized in H. Suzuki (1994) and in N. R. Thomas (1994).

EXAMPLE XV NMR Characterization of Fn3 and Comparison of the Fn3Secreted by Yeast With That Secreted by E. coli

[0201] Nuclear magnetic resonance (NMR) experiments are performed toidentify the contact surface between FnAb and a target molecule, e.g.,monobodies to fluorescein, ubiquitin, RNaseA and soluble derivatives ofdigoxigenin. The information is then be used to improve the affinity andspecificity of the monobody. Purified monobody samples are dissolved inan appropriate buffer for NMR spectroscopy using Amicon ultrafiltrationcell with a YM-3 membrane. Buffers are made with 90% H₂O/10% D₂O(distilled grade, Isotec) or with 100% D₂O. Deuterated compounds (e.g.acetate) are used to eliminate strong signals from them.

[0202] NMR experiments are performed on a Varian Unity INOVA 600spectrometer equipped with four RF channels and a triple resonance probewith pulsed field gradient capability. NMR spectra are analyzed usingprocessing programs such as Felix (Molecular Simulations), nmrPipe,PIPP, and CAPP (Garrett, et al., 1991; Delaglio, et al., 1995) on UNIXworkstations. Sequence specific resonance assignments are made usingwell-established strategy using a set of triple resonance experiments(CBCA(CO)NH and HNCACB) (Grzesiek & Bax, 1992; Wittenkind & Mueller,1993).

[0203] Nuclear Overhauser effect (NOE) is observed between ¹H nucleicloser than approximately 5 Å, which allows one to obtain information oninterproton distances. A series of double- and triple-resonanceexperiments (Table 6; for recent reviews on these techniques, see Bax &Grzesiek, 1993 and Kay, 1995) are performed to collect distance (i.e.NOE) and dihedral angle (J-coupling) constraints. Isotope-filteredexperiments are performed to determine resonance assignments of thebound ligand and to obtain distance constraints within the ligand andthose between FnAb and the ligand. Details of sequence specificresonance assignments and NOE peak assignments have been described indetail elsewhere (Clore & Gronenborn, 1991; Pascal, et al., 1994b;Metzler, et al., 1996). TABLE 6 NMR experiments for structurecharacterization Experiment Name Reference 1. reference spectra 2D-¹H,¹⁵N-HSQC (Bodenhausen & Ruben, 1980; Kay, et al., 1992) 2D-¹H, ¹³C-HSQC(Bodenhausen & Ruben, 1980; Vuister & Bax, 1992) 2. backbone and sidechain resonance assignments of ¹³C/¹⁵N-labeled protein 3D-CBCA(CO)NH(Grzesiek & Bax, 1992) 3D-HNCACB (Wittenkind & Mueller, 1993) 3D-C(CO)NH(Logan et al., 1992; Grzesiek et al., 1993) 3D-H(CCO)NH3D-HBHA(CBCACO)NH (Grzesiek & Bax, 1993) 3D-HCCH-TOCSY (Kay et al.,1993) 3D-HCCH-COSY (Ikura et al., 1991) 3D-¹H, ¹⁵N-TOCSY-HSQC (Zhang etal., 1994) 2D-HB(CBCDCE)HE (Yamazaki et al., 1993) 3. resonanceassignments of unlabeled ligand 2D-isotope-filtered ¹H-TOCSY2D-isotope-filtered ¹H-COSY 2D-isotope-filtered ¹H-NOESY (Ikura & Bax,1992) 4. structural constraints within labeled protein 3D-¹H,¹⁵N-NOESY-HSQC (Zhang et al., 1994) 4D-¹H, ¹³C-HMQC-NOESY-HMQC (Vuisteret al., 1993) 4D-¹H, ¹³C, ¹⁵N-HSQC-NOESY-HSQC (Muhandiram et al., 1993;Pascal et al., 1994a) within unlabeled ligand 2D-isotope-filtered¹H-NOESY (Ikura & Bax, 1992) interactions between protein and ligand3D-isotope-filtered ¹H, ¹⁵N-NOESY-HSQC 3D-isotope-filtered ¹H,¹³C-NOESY-HSQC (Lee et al., 1994) 5. dihedral angle constraintsJ-molulated ¹H, ¹⁵N-HSQC (Billeter et al., 1992) 3D-HNHB (Archer et al.,1991)

[0204] Backbone ¹H, ¹⁵N and ¹³C resonance assignments for a monobody arecompared to those for wild-type Fn3 to assess structural changes in themutant. Once these data establish that the mutant retains the globalstructure, structural refinement is performed using experimental NOEdata. Because the structural difference of a monobody is expected to beminor, the wild-type structure can be used as the initial model aftermodifying the amino acid sequence. The mutations are introduced to thewild-type structure by interactive molecular modeling, and then thestructure is energy-minimized using a molecular modeling program such asQuanta (Molecular Simulations). Solution structure is refined usingcycles of dynamical simulated annealing (Nilges et al., 1988) in theprogram X-PLOR (Brünger, 1992). Typically, an ensemble of fiftystructures is calculated. The validity of the refined structures isconfirmed by calculating a fewer number of structures from randomlygenerated initial structures in X-PLOR using the YASAP protocol (Nilges,et al., 1991). Structure of a monobody-ligand complex is calculated byfirst refining both components individually using intramolecular NOEs,and then docking the two using intermolecular NOEs.

[0205] For example, the ¹H, ¹⁵N-HSQC spectrum for thefluorescein-binding monobody LB25.5 is shown in FIG. 14. The spectrumshows a good dispersion (peaks are spread out) indicating that LB25.5 isfolded into a globular conformation. Further, the spectrum resemblesthat for the wild-type Fn3, showing that the overall structure of LB25.5is similar to that of Fn3. These results demonstrate that ligand-bindingmonobodies can be obtained without changing the global fold of the Fn3scaffold.

[0206] Chemical shift perturbation experiments are performed by formingthe complex between an isotope-labeled FnAb and an unlabeled ligand. Theformation of a stoichiometric complex is followed by recording the HSQCspectrum. Because chemical shift is extremely sensitive to nuclearenvironment, formation of a complex usually results in substantialchemical shift changes for resonances of amino acid residues in theinterface. Isotope-edited NMR experiments (2D HSQC and 3D CBCA(CO)NH)are used to identify the resonances that are perturbed in the labeledcomponent of the complex; i.e. the monobody. Although the possibility ofartifacts due to long-range conformational changes must always beconsidered, substantial differences for residues clustered on continuoussurfaces are most likely to arise from direct contacts (Chen et al.,1993; Gronenborn & Clore, 1993).

[0207] An alternative method for mapping the interaction surfaceutilizes amide hydrogen exchange (HX) measurements. HX rates for eachamide proton are measured for ¹⁵N labeled monobody both free andcomplexed with a ligand. Ligand binding is expected to result indecreased amide HX rates for monobody residues in the interface betweenthe two proteins, thus identifying the binding surface. HX rates formonobodies in the complex are measured by allowing HX to occur for avariable time following transfer of the complex to D₂O; the complex isdissociated by lowering pH and the HSQC spectrum is recorded at low pHwhere amide HX is slow. Fn3 is stable and soluble at low pH, satisfyingthe prerequisite for the experiments.

EXAMPLE XVI Construction and Analysis of Fn3-Display System Specific forUbiquitin

[0208] An Fn3-display system was designed and synthesized,ubiquitin-binding clones were isolated and a major Fn3 mutant in theseclones was biophysically characterized.

[0209] Gene construction and phage display of Fn3 was performed as inExamples I and II above. The Fn3-phage pIII fusion protein was expressedfrom a phagemid-display vector, while the other components of the M13phage, including the wild-type pIII, were produced using a helper phage(Basset al., 1990). Thus, a phage produced by this system should containless than one copy of Fn3 displayed on the surface. The surface displayof Fn3 on the phage was detected by ELISA using an anti-Fn3 antibody.Only phages containing the Fn3-pIII fusion vector reacted with theantibody.

[0210] After confirming the phage surface to display Fn3, a phagedisplay library of Fn3 was constructed as in Example III. Randomsequences were introduced in the BC and FG loops. In the first library,five residues (77-81) were randomized and three residues (82-84) weredeleted from the FG loop. The deletion was intended to reduce theflexibility and improve the binding affinity of the FG loop. Fiveresidues (26-30) were also randomized in the BC loop in order to providea larger contact surface with the target molecule. Thus, the resultinglibrary contains five randomized residues in each of the BC and FG loops(Table 7). This library contained approximately 10⁸ independent clones.

[0211] Library Screening

[0212] Library screening was performed using ubiquitin as the targetmolecule. In each round of panning, Fn3-phages were absorbed to aubiquitin-coated surface, and bound phages were eluted competitivelywith soluble ubiquitin. The recovery ratio improved from 4.3×10⁻⁷ in thesecond round to 4.5×10⁻⁶ in the fifth round, suggesting an enrichment ofbinding clones. After five founds of panning, the amino acid sequencesof individual clones were determined (Table 7). TABLE 7 Sequences in thevariegated loops of enriched clones Name BC loop FG loop Frequency WildGCAGTTACCGTGCGT GGCCGTGGTGACAGCCCAGCGAGC — Type (SEQ ID NO:93) (SEQ IDNO:95) AlaValThrValArg GlyArgGlyAspSerProAlaSer (SEQ ID NO:94) (SEQ IDNO:96) Library^(a) NNKNNKNNKNNKNNK NNKNNKNNKNNKNNK--------- — XXXXXXXXXX(deletion) clone1 TCGAGGTTGCGGCGG CCGCCGTGGAGGGTG 9 (Ubi4) (SEQ IDNO:97) (SEQ ID NO:99) SerArgLeuArgArg ProProTrpArgVal (SEQ ID NO:98)(SEQ ID NO:100) clone2 GGTCAGCGAACTTTT AGGCGGTGGTGGGCT 1 (SEQ ID NO:101)(SEQ ID NO:103) GlyGlnArgThrPhe ArgArgTrpTrpAla (SEQ ID NO:102) (SEQ IDNO:104) clone3 GCGAGGTGGACGCTT AGGCGGTGGTGGTGG 1 (SEQ ID NO:105) (SEQ IDNO:107) AlaArgTrpThrLeu ArgArgTrpTrpTrp (SEQ ID NO:106) (SEQ ID NO:108)

[0213] A clone, dubbed Ubi4, dominated the enriched pool of Fn3variants. Therefore, further investigation was focused on this Ubi4clone. Ubi4 contains four mutations in the BC loop (Arg 30 in the BCloop was conserved) and five mutations and three deletions in the FGloop. Thus 13% (12 out of 94) of the residues were altered in Ubi4 fromthe wild-type sequence.

[0214]FIG. 15 shows a phage ELISA analysis of Ubi4. The Ubi4 phage bindsto the target molecule, ubiquitin, with a significant affinity, while aphage displaying the wild-type Fn3 domain or a phase with no displayedmolecules show little detectable binding to ubiquitin (FIG. 15a). Inaddition, the Ubi4 phage showed a somewhat elevated level of backgroundbinding to the control surface lacking the ubiquitin coating. Acompetition ELISA experiments shows the IC₅₀ (concentration of the freeligand which causes 50% inhibition of binding) of the binding reactionis approximately 5 μM (FIG. 15b). BSA, bovine ribonuclease A andcytochrome C show little inhibition of the Ubi4-ubiquitin bindingreaction (FIG. 15c), indicating that the binding reaction of Ubi4 toubiquitin does result from specific binding.

[0215] Characterization of a Mutant Fn3 Protein

[0216] The expression system yielded 50-100 mg Fn3 protein per literculture. A similar level of protein expression was observed for the Ubi4clone and other mutant Fn3 proteins.

[0217] Ubi4-Fn3 was expressed as an independent protein. Though amajority of Ubi4 was expressed in E. coli as a soluble protein, itssolubility was found to be significantly reduced as compared to that ofwild-type Fn3. Ubi4 was soluble up to ˜20 μM at low pH, with much lowersolubility at neutral pH. This solubility was not high enough fordetailed structural characterization using NMR spectroscopy or X-raycrystallography.

[0218] The solubility of the Ubi4 protein was improved by adding asolubility tail, GKKGK (SEQ ID NO:109), as a C-terminal extension. Thegene for Ubi4-Fn3 was subcloned into the expression vector pAS45 usingPCR. The C-terminal solubilization tag, GKKGK (SEQ ID NO:109), wasincorporated in this step. E. coli BL21 (DE3) (Novagen) was transformedwith the expression vector (pAS45 and its derivatives). Cells were grownin M9 minimal media and M9 media supplemented with Bactotryptone (Difco)containing ampicillin (200 μg/ml). For isotopic labeling, ¹⁵N NH₄Clreplaced unlabeled NH₄Cl in the media. 500 ml medium in a 2 liter baffleflask was inoculated with 10 ml of overnight culture and agitated at 37°C. IPTG was added at a final concentration of 1 mM to initiate proteinexpression when OD (600 nm) reaches one. The cells were harvested bycentrifugation 3 hours after the addition of IPTG and kept frozen at−70° C. until used.

[0219] Proteins were purified as follows. Cells were suspended in 5ml/(g cell) of Tris (50 mM, pH 7.6) containing phenylmethylsulfonylfluoride (1 mM). Hen egg lysozyme (Sigma) was added to a finalconcentration of 0.5 mg/ml. After incubating the solution for 30 minutesat 37° C., it was sonicated three times for 30 seconds on ice. Celldebris was removed by centrifugation. Concentrated sodium chloride wasadded to the solution to a final concentration of 0.5 M. The solutionwas applied to a Hi-Trap chelating column (Pharmacia) preloaded withnickel and equilibrated in the Tris buffer containing sodium chloride(0.5 M). After washing the column with the buffer, histag-Fn3 was elutedwith the buffer containing 500 mM imidazole. The protein was furtherpurified using a ResourceS column (Pharmacia) with a NaCl gradient in asodium acetate buffer (20 mM, pH 4.6).

[0220] With the GKKGK (SEQ ID NO: 109) tail, the solubility of the Ubi4protein was increased to over 1 mM at low pH and up to ˜50 μM at neutralpH. Therefore, further analyses were performed on Ubi4 with thisC-terminal extension (hereafter referred to as Ubi4-K). It has beenreported that the solubility of a minibody could be significantlyimproved by addition of three Lys residues at the N- or C-termini(Bianchi et al., 1994). In the case of protein Rop, a non-structuredC-terminal tail is critical in maintaining its solubility (Smith et al.,1995).

[0221] Oligomerization states of the Ubi4 protein were determined usinga size exclusion column. The wild-type Fn3 protein was monomeric at lowand neutral pH's. However, the peak of the Ubi4-K protein wassignificantly broader than that of wild-type Fn3, and eluted after thewild-type protein. This suggests interactions between Ubi4-K and thecolumn material, precluding the use of size exclusion chromatography todetermine the oligomerization state of Ubi4. NMR studies suggest thatthe protein is monomeric at low pH.

[0222] The Ubi4-K protein retained a binding affinity to ubiquitin asjudged by ELISA (FIG. 15d). However, an attempt to determine thedissociation constant using a biosensor (Affinity Sensors, Cambridge,U.K.) failed because of high background binding of Ubi4-K-Fn3 to thesensor matrix. This matrix mainly consists of dextran, consistent withthe observation that interactions between Ubi4-K interacts with thecross-linked dextran of the size exclusion column.

EXAMPLE XVII Stability Measurements of Monobodies

[0223] Guanidine hydrochloride (GuHCl)-induced unfolding and refoldingreactions were followed by measuring tryptophan fluorescence.Experiments were performed on a Spectronic AB-2 spectrofluorometerequipped with a motor-driven syringe (Hamilton Co.). The cuvettetemperature was kept at 30° C. The spectrofluorometer and the syringewere controlled by a single computer using a home-built interface. Thissystem automatically records a series of spectra following GuHCltitration. An experiment started with a 1.5 ml buffer solutioncontaining 5 μM protein. An emission spectrum (300-400 nm; excitation at290 nm) was recorded following a delay (3-5 minutes) after eachinjection (50 or 100 μl) of a buffer solution containing GuHCl. Thesesteps were repeated until the solution volume reached the full capacityof a cuvette (3.0 ml). Fluorescence intensities were normalized asratios to the intensity at an isofluorescent point which was determinedin separate experiments. Unfolding curves were fitted with a two-statemodel using a nonlinear least-squares routine (Santoro & Bolen, 1988).No significant differences were observed between experiments with delaytimes (between an injection and the start of spectrum acquisition) of 2minutes and 10 minutes, indicating that the unfolding/refoldingreactions reached close to an equilibrium at each concentration pointwithin the delay times used.

[0224] Conformational stability of Ubi4-K was measured usingabove-described GuHCl-induced unfolding method. The measurements wereperformed under two sets of conditions; first at pH 3.3 in the presenceof 300 mM sodium chloride, where Ubi4-K is highly soluble, and second inTBS, which was used for library screening. Under both conditions, theunfolding reaction was reversible, and we detected no signs ofaggregation or irreversible unfolding. FIG. 16 shows unfoldingtransitions of Ubi4-K and wild-type Fn3 with the N-terminal (his)₆ tagand the C-terminal solubility tag. The stability of wild-type Fn3 wasnot significantly affected by the addition of these tags. Parameterscharacterizing the unfolding transitions are listed in Table 8. TABLE 8Stability parameters for Ubi4 and wild-type Fn3 as determined byGuHCl-induced unfolding Protein ΔG₀ (kcal mol⁻¹) m_(G) (kcal mol⁻¹ M⁻¹)Ubi4 (pH 7.5) 4.8 ± 0.1 2.12 ± 0.04 Ubi4 (pH 3.3) 6.5 ± 0.1 2.07 ± 0.02Wild-type (pH 7.5) 7.2 ± 0.2 1.60 ± 0.04 Wild-type (pH 3.3) 11.2 ± 0.1 2.03 ± 0.02

[0225] Though the introduced mutations in the two loops certainlydecreased the stability of Ubi4-K relative to wild-type Fn3, thestability of Ubi4 remains comparable to that of a “typical” globularprotein. It should also be noted that the stabilities of the wild-typeand Ubi4-K proteins were higher at pH 3.3 than at pH 7.5.

[0226] The Ubi4 protein had a significantly reduced solubility ascompared to that of wild-type Fn3, but the solubility was improved bythe addition of a solubility tail. Since the two mutated loops includethe only differences between the wild-type and Ubi4 proteins, theseloops must be the origin of the reduced solubility. At this point, it isnot clear whether the aggregation of Ubi4-K is caused by interactionsbetween the loops, or by interactions between the loops and theinvariable regions of the Fn3 scaffold.

[0227] The Ubi4-K protein retained the global fold of Fn3, showing thatthis scaffold can accommodate a large number of mutations in the twoloops tested. Though the stability of the Ubi4-K protein issignificantly lower than that of the wild-type Fn3 protein, the Ubi4protein still has a conformational stability comparable to those forsmall globular proteins. The use of a highly stable domain as a scaffoldis clearly advantageous for introducing mutations without affecting theglobal fold of the scaffold. In addition, the GuHCl-induced unfolding ofthe Ubi4 protein is almost completely reversible. This allows thepreparation of a correctly folded protein even when a Fn3 mutant isexpressed in a misfolded form, as in inclusion bodies. The modeststability of Ubi4 in the conditions used for library screening indicatesthat Fn3 variants are folded on the phage surface. This suggests that aFn3 clone is selected by its binding affinity in the folded form, not ina denatured form. Dickinson et al proposed that Val 29 and Arg 30 in theBC loop stabilize Fn3. Val 29 makes contact with the hydrophobic core,and Arg 30 forms hydrogen bonds with Gly 52 and Val 75. In Ubi4-Fn3, Val29 is replaced with Arg, while Arg 30 is conserved. The FG loop was alsomutated in the library. This loop is flexible in the wild-typestructure, and shows a large variation in length among human Fn3 domains(Main et al., 1992). These observations suggest that mutations in the FGloop may have less impact on stability. In addition, the N-terminal tailof Fn3 is adjacent to the molecular surface formed by the BC and FGloops (FIGS. 1 and 17) and does not form a well-defined structure.Mutations in the N-terminal tail would not be expected to have strongdetrimental effects on stability. Thus, residues in the N-terminal tailmay be good sites for introducing additional mutations.

EXAMPLE XVIII NMR Spectroscopy of Ubi4-Fn3

[0228] Ubi4-Fn3 was dissolved in [²H]-Gly HCl buffer (20 mM, pH 3.3)containing NaCl (300 mM) using an Amicon ultrafiltration unit. The finalprotein concentration was 1 mM. NMR experiments were performed on aVarian Unity INOVA 600 spectrometer equipped with a triple-resonanceprobe with pulsed field gradient. The probe temperature was set at 30°C. HSQC, TOCSY-HSQC and NOESY-HSQC spectra were recorded using publishedprocedures (Kay et al., 1992; Zhang et al., 1994). NMR spectra wereprocessed and analyzed using the NMRPipe and NMRView software (Johnson &Blevins, 1994; Delaglio et al., 1995) on UNIX workstations.Sequence-specific resonance assignments were made using standardprocedures (Wüthrich, 1986; Clore & Gronenborn, 1991). The assignmentsfor wild-type Fn3 (Baron et al., 1992) were confirmed using a¹⁵N-labeled protein dissolved in sodium acetate buffer (50 mM, pH 4.6)at 30° C.

[0229] The three-dimensional structure of Ubi4-K was characterized usingthis heteronuclear NMR spectroscopy method. A high quality spectrumcould be collected on a 1 mM solution of ¹⁵N-labeled Ubi4 (FIG. 17a) atlow pH. The linewidth of amide peaks of Ubi4-K was similar to that ofwild-type Fn3, suggesting that Ubi4-K is monomeric under the conditionsused. Complete assignments for backbone ¹H and ¹⁵N nuclei were achievedusing standard 1H, ¹⁵N double resonance techniques, except for a row ofHis residues in the N-terminal (His)₆ tag. There were a few weak peaksin the HSQC spectrum which appeared to originate from a minor speciescontaining the N-terminal Met residue. Mass spectroscopy analysis showedthat a majority of Ubi4-K does not contain the N-terminal Met residue.FIG. 17 shows differences in ¹HN and ¹⁵N chemical shifts between Ubi4-Kand wild-type Fn3. Only small differences are observed in the chemicalshifts, except for those in and near the mutated BC and FG loops. Theseresults clearly indicate that Ubi4-K retains the global fold of Fn3,despite the extensive mutations in the two loops. A few residues in theN-terminal region, which is close to the two mutated loops, also exhibitsignificant chemical differences between the two proteins. An HSQCspectrum was also recorded on a 50 μM sample of Ubi4-K in TBS. Thespectrum was similar to that collected at low pH, indicating that theglobal conformation of Ubi4 is maintained between pH 7.5 and 3.3.

EXAMPLE XIX Stabilization of Fn3 Domain by Removing UnfavorableElectrostatic Interactions on the Protein Surface

[0230] Introduction

[0231] Increasing the conformational stability of a protein by mutationis a major interest in protein design and biotechnology. Thethree-dimensional structures of proteins are stabilized by combinationof different types of forces. The hydrophobic effect, van der Waalsinteractions and hydrogen bonds are known to contribute to stabilize thefolded state of proteins (Kauzmann, W. (1959) Adv. Prot. Chem. 14, 1-63;Dill, K. A. (1990) Biochemistry 29, 7133-7155; Pace, C. N., Shirley, B.A., McNutt, M. & Gajiwala, K. (1996) Faseb J 10, 75-83). Thesestabilizing forces primarily originate from residues that are wellpacked in a protein, such as those that constitute the hydrophobic core.Because a change in the protein core would induce a rearrangement ofadjacent moieties, it is difficult to improve protein stability byincreasing these forces without massive computation (Malakauskas, S. M.& Mayo, S. L. (1998) Nat Struct Biol 5, 470-475). Ion pairs betweencharged groups are commonly found on the protein surface (Creighton, T.E. (1993) Proteins: structures and molecular properties, Freeman, N.Y.),and an ion pair could be introduced to a protein with small structuralperturbations. However, a number of studies have demonstrated that theintroduction of an attractive electrostatic interaction, such as an ionpair, on protein surface has small effects on stability (Dao-pin, S.,Sauer, U., Nicholson, H. & Matthews, B. W. (1991) Biochemistry 30,7142-7153; Sali, D., Bycroft, M. & Fersht, A. R. (1991) J. Mol. Biol.220, 779-788). A large desolvation penalty and the loss ofconformational entropy of amino acid side chains oppose the favorableelectrostatic contribution (Yang, A.-S. & Honig, B. (1992) Curr. Opin.Struct. Biol. 2, 40-45; Hendsch, Z. S. & Tidor, B. (1994) Protein Sci.3, 211-226). Recent studies demonstrated that repulsive electrostaticinteractions on the protein surface, in contrast, may significantlydestabilize a protein, and that it is possible to improve proteinstability by optimizing surface electrostatic interactions (Loladze, V.V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999)Biochemistry 38, 16419-16423; Perl, D., Mueller, U., Heinemann, U. &Schmid, F. X. (2000) Nat Struct Biol 7, 380-383; Spector, S., Wang, M.,Carp, S. A., Robblee, J., Hendsch, Z. S., Fairman, R., Tidor, B. &Raleigh, D. P. (2000) Biochemistry 39, 872-879; Grimsley, G. R., Shaw,K. L., Fee, L. R., Alston, R. W., Huyghues-Despointes, B. M., Thurlkill,R. L., Scholtz, J. M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849). Inthe present experiments, the inventor improved protein stability bymodifying surface electrostatic interactions.

[0232] During the characterization of monobodies it was found that theseproteins, as well as wild-type FNfn10, are significantly more stable atlow pH than at neutral pH (Koide, A., Bailey, C. W., Huang, X. & Koide,S. (1998) J. Mol Biol. 284, 1141-1151). These observations indicate thatchanges in the ionization state of some moieties in FNfn10 modulate theconformational stability of the protein, and suggest that it might bepossible to enhance the conformational stability of FNfn10 at neutral pHby adjusting electrostatic properties of the protein. Improving theconformational stability of FNfn10 will also have practical importancein the use of FNfn10 as a scaffold in biotechnology applications.

[0233] Described below are experiments that detailed characterization ofthe pH dependence of FNfn10 stability, identified unfavorableinteractions between side chain carboxyl groups, and improved theconformational stability of FNfn10 by point mutations on the surface.The results demonstrate that the surface electrostatic interactionscontribute significantly to protein stability, and that it is possibleto enhance protein stability by rationally modulating theseinteractions.

[0234] Experimental Procedures

[0235] Protein expression and purification

[0236] The wild-type protein used for the NMR studies contained residues1-94 of FNfn10 (residue numbering is according to FIG. 2(a) of Koide etal. (Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol.Biol. 284, 1141-1151)), and additional two residues (Met-Gln) at theN-terminus (these two residues are numbered −2 and −1, respectively).The gene coding for the protein was inserted in pET3a (Novagen, Wis.).Eschericha coli BL21 (DE3) transformed with the expression vector wasgrown in the M9 minimal media supplemented with ¹³C-glucose and¹⁵N-ammonium chloride (Cambridge Isotopes) as the sole carbon andnitrogen sources, respectively. Protein expression was induced asdescribed previously (Koide, A., Bailey, C. W., Huang, X. & Koide, S.(1998) J. Mol. Biol. 284, 1141-1151). After harvesting the cells bycentrifuge, the cells were lysed as described (Koide, A., Bailey, C. W.,Huang, X. & Koide, S. (1998) J. Mol. Biol. 284, 1141-1151). Aftercentrifugation, supernatant was dialyzed against 10 mM sodium acetatebuffer (pH 5.0), and the protein solution was applied to a SP-SepharoseFastFlow column (Amersham Pharmacia Biotech), and FN3 was eluted with agradient of sodium chloride. The protein was concentrated using anAmicon concentrator using YM-3 membrane (Millipore).

[0237] The wild-type protein used for the stability measurementscontained an N-terminal histag (MGSSHHHHHHSSGLVPRGSH) (SEQ ID NO:114)and residues-2-94 of FNfn10. The gene for FN3 described above wasinserted in pET15b (Novagen). The protein was expressed and purified asdescribed (Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J.Mol. Biol. 284, 1141-1151). The wild-type protein used for measurementsof the pH dependence shown in FIG. 22 contained Arg 6 to Thr mutation,which had originally been introduced to remove a secondary thrombincleavage site (Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J.Mol. Biol. 284, 1141-1151). Because Asp 7, which is adjacent to Arg 6,was found to be critical in the pH dependence of FN3 stability asdetailed under Results, subsequent studies were performed using thewild-type, Arg 6, background. The genes for the D7N and D7K mutants wereconstructed using standard polymerase chain reactions, and inserted inpET15b. These proteins were prepared in the same manner as for thewild-type protein. ¹³C, ¹⁵N-labeled proteins for pK_(a) measurementswere prepared as described above, and the histag moiety was not removedfrom these proteins.

[0238] Chemical denaturation measurements

[0239] Proteins were dissolved to a final concentration of 5 μM in 10 mMsodium citrate buffer at various pH containing 100 mM sodium chloride.Guanidine HCl (GuHCl)-induce unfolding experiments were performed asdescribed previously (Koide, A., Bailey, C. W., Huang, X. & Koide, S.(1998) J. Mol. Biol. 284, 1141-1151; Koide, S., Bu, Z., Risal, D., Pham,T.-N., Nakagawa, T., Tamura, A. & Engelman, D. M. (1999) Biochemistry38, 4757-4767). GuHCl concentration was determined using an Abberefractometer (Spectronic Instruments) as described (Pace, C. N. &Sholtz, J. M. (1997) in Protein structure. A practical approach(Creighton, T. E., Ed.) Vol. pp 299-321, IRL Press, Oxford). Data wereanalyzed according to the two-state model as described (Koide, A.,Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol. 284,1141-1151; Santoro, M. M. & Bolen, D. W. (1988) Biochemistry 27,8063-8068.).

[0240] Thermal denaturation measurements

[0241] Proteins were dissolved to a final concentration of 5 μM in 20 mMsodium phosphate buffer (pH 7.0) containing 0.1 or 1 M sodium chlorideor in 20 mM glycine HCl buffer (pH 2.4) containing 0.1 or 1 M sodiumchloride. Additionally 6.3 M urea was included in all solutions toensure reversibility of the thermal denaturation reaction. In theabsence of urea it was found that denatured FNfn10 adheres to quartzsurface, and that the thermal denaturation reaction was irreversible.Circular dichroism measurements were performed using a Model 202spectrometer equipped with a Peltier temperature controller (AvivInstruments). A cuvette with a 0.5-cm pathlength was used. Theellipticity at 227 nm was recorded as the sample temperature was raisedat a rate of approximately 1 ° C. per minute. Because of decompositionof urea at high temperature, the pH of protein solutions tended to shiftupward during an experiment. The pH of protein solution was measuredbefore and after each 30 thermal denaturation measurement to ensure thata shift no more than 0.2 pH unit occurred in each measurement. At pH2.4, two sections of a thermal denaturation curve (30-65° C. and 60-95°C.) were acquired from separate samples, in order to avoid a large pHshift. The thermal denaturation data were fit with the standardtwo-state model (Pace, C. N. & Sholtz, J. M. (1997) in Proteinstructure. A practical approach (Creighton, T. E., Ed.) Vol. pp 299-321,IRL Press, Oxford):

ΔG(T)=ΔH _(m)(1−T/T _(m))−ΔC _(p)[(T _(m) −T)+T1n(T/T _(m))]

[0242] where ΔG(T) is the Gibbs free energy of unfolding at temperatureT, ΔH_(m) is the enthalpy change upon unfolding at the midpoint of thetransition, T_(m), and ΔC_(p) is the heat capacity change uponunfolding. The value for ΔC_(p) was fixed at 1.74 kcal mol⁻¹ K⁻¹,according to the approximation of Myers et al. (Myers, J. K., Pace, C.N. & Scholtz, J. M. (1995) Protein Sci. 4, 2138-2148). Most of thedatasets taken in the presence of 1 M NaCl did not have a sufficientbaseline for the unfolded state, and thus it was assumed the slope ofthe unfolded baseline in the presence of 1 M NaCl to be identical tothat determined in the presence of 0.1 M NaCl.

[0243] NMR spectroscopy

[0244] NMR experiments were performed at 30° C. on an INOVA 600spectrometer (Varian Instruments). The C(CO)NH experiment (Grzesiek, S.,Anglister, J. & Bax, A. (1993) J. Magn. Reson. B 101, 114-119) and theCBCACOHA experiment (Kay, L. E. (1993) J. Am. Chem. Soc. 115, 2055-2057)were collected on a [¹³C, ¹⁵N]-wild-type FNfn10 sample (1 mM) dissolvedin 50 mM sodium acetate buffer (pH 4.6) containing 5% (v/v) deuteriumoxide, using a Varian 5 mm triple resonance probe with pulsed fieldgradient. The carboxyl ¹³C resonances were assigned based on thebackbone ¹H, ¹³C and ¹⁵N resonance assignments of FNfn10 (Baron, M.,Main, A. L., Driscoll, P. C., Mardon, H. J., Boyd, J. & Campbell, I. D.(1992) Biochemistry 31, 2068-2073). pH titration of carboxyl resonanceswere performed on a 0.3 mM FNfn10 sample dissolved in 10 mM sodiumcitrate containing 100 mM sodium chloride and 5% (v/v) deuterium oxide.An 8 mm triple-resonance, pulse-field gradient probe (NanolacCorporation) was used for pH titration. Two-dimensional H(C)CO spectrawere collected using the CBCACOHA pulse sequence as described previously(McIntosh, L. P., Hand, G., Johnson, P. E., Joshi, M. D., Koerner, M.,Plesniak, L. A., Ziser, L., Wakarchuk, W. W. & Withers, S. G. (1996)Biochemistry 35, 9958-9966). Sample pH was changed by adding smallaliquots of hydrochloric acid, and pH was measured before and aftertaking NMR data. ¹H, ¹⁵N-HSQC spectra were taken as described previously(Kay, L. E., Keifer, P. & Saarinen, T. (1992) J. Am. Chem. Soc. 114,10663-10665). NMR data were processed using the NMRPipe package(Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. & Bax,A. (1995) J. Biomol. NMR 6, 277-293), and analyzed using the NMRViewsoftware (Johnson, B. A. & Blevins, R. A. (1994) J. Biomol. NMR 4,603-614).

[0245] NMR titration curves of the carboxyl ¹³C resonances were fit tothe Henderson-Hasselbalch equation to determine pK_(a)'s:

δ(pH)=(δ_(acid)+δ_(base)10^((pH−pK) ^(_(a)) ⁾)/(1+10^((pH−pKa)))

[0246] where δ is the measured chemical shift, δ_(acid) is the chemicalshift associated with the protonated state, δ_(base) is the chemicalshift associated with the deprotonated state, and pK_(a) is the pK_(a)value for the residue. Data were also fit to an equation with twoionizable groups:

δ(pH)=(δ_(AH2′)+δ_(AH)10^((pH−pK) ^(_(a1)) ⁾+δ_(A)10^((2pH−pK) ^(_(a1))^(−pK) ^(_(a2)) ⁾)/(1+10^((pH−pK) ^(_(a1)) ⁾⁺10^((2pH−pK) ^(_(a1))^(−pK) ^(_(a2)) ⁾)

[0247] where δ_(AH2), δ_(AH) and δ_(A) are the chemical shiftsassociated with the fully protonated, singularly protonated anddeprotonated states, respectively, and pK_(a1) and pK_(a2) are pK_(a)'sassociated with the two ionization steps. Data fitting was performedusing the nonlinear least-square regression method in the program IgorPro (WaveMetrix, Oreg.) on a Macintosh computer.

[0248] Results

[0249] pH Dependence of FNfn10 stability

[0250] Previously, it was found that FNfn10 is more stable at acidic pHthan at neutral pH (Koide, A., Bailey, C. W., Huang, X. & Koide, S.(1998) J. Mol. Biol. 284, 1141-1151). In the present experiments, the pHdependence of its stability was further characterized. Because of itshigh stability, FNfn10 could not be fully denatured in urea at 30° C.Thus GuHCl-induced chemical denaturation (FIG. 18) was used. Thedenaturation reaction was fully reversible under all conditions tested.In order to minimize errors caused by extrapolation, the free energy ofunfolding at 4 M GuHCl was used for comparison (FIG. 18). The stabilityincreased as the pH was lowered, with apparent plateaus at both ends ofthe pH range. The pH dependence curve has an apparent transitionmidpoint near pH 4. In addition, a gradual increase in the m value, thedependence of the unfolding free energy on denaturant concentration wasnoted. Pace et al. reported a similar pH dependence of the m value forbarnase (Pace, C. N., Laurents, D. V. & Erickson, R. E. (1992)Biochemistry 31, 2728-2734). These results indicate that FNfn10 containsinteractions that stabilize the protein at low pH, or those thatdestabilize it at neutral pH. The results also suggest that byidentifying and altering the interactions that give rise to the pHdependence, one may be able to improve the stability of FNfn10 atneutral pH to a degree similar to that found at low pH.

[0251] Determination of pKa's of the side chain carboxyl groups inwild-type FNfn10

[0252] The pH dependence of FNfn10 stability suggests that amino acidswith pK_(a) near 4 are involved in the observed transition. The carboxylgroups of Asp and Glu generally have pK_(a) in this range (Creighton, T.E. (1993) Proteins: structures and molecular properties, Freeman, N.Y.).It is well known that if a carboxyl group has unfavorable (i.e.destabilizing) interactions in the folded state, its pK_(a) is shiftedto a higher value from its unperturbed value (Yang, A.-S. & Honig, B.(1992) Curr. Opin. Struct. Biol. 2, 40-45). If a carboxyl group hasfavorable interactions in the folded state, it has a lower pK_(a). Thus,the pK_(a) values of all carboxylates in FNfn10 using heteronuclear NMRspectroscopy were determined in order to identify stabilizing anddestabilizing interactions involving carboxyl groups.

[0253] First, the ¹³C resonance for the carboxyl carbon of each Asp andGlu residue in FN3 was assigned (FIG. 19). Next, pH titration of the ¹³Cresonances for these groups was performed (FIG. 20). Titration curvesfor Asp 3, 67 and 80, and Glu 38 and 47 could be fit well with theHenderson-Hasselbalch equation with a single pK_(a). The pK_(a) valuesfor these residues (Table 9) are either close to or slightly lower thantheir respective unperturbed values (3.8-4.1 for Asp, and 4.1-4.6 forGlu (Kuhlman, B., Luisi, D. L., Young, P. & Raleigh, D. P. (1999)Biochemistry 38, 4896-4903)), indicating that these carboxyl groups areinvolved in neutral or slightly favorable electrostatic interactions inthe folded state. TABLE 9 pK_(a) values for Asp and Glu residues inFN3¹. Protein Residue Wild-Type D7N D7K E9 3.84, 5.40² 4.98 4.53 E383.79 3.87 3.86 E47 3.94 3.99 3.99 D3 3.66 3.72 3.74 D7 3.54, 5.54² — —D23 3.54, 5.25² 3.68 3.82 D67 4.18 4.17 4.14 D80 3.40 3.49 3.48

[0254] The titration curves for Asp 7 and 23, and Glu 9 were fit betterwith the Henderson-Hasselbalch equation with two pK_(a) values, and oneof the two pK_(a) values for each were shifted higher than therespective unperturbed values (FIG. 19B). The titration curves with twoapparent pK_(a) values of these carboxyl groups may be due to influenceof an ionizable group in the vicinity. In the three-dimensionalstructure of FNfn10 (Main, A. L., Harvey, T. S., Baron, M., Boyd, J. &Campbell, I. D. (1992) Cell 71, 671-678), Asp 7 and 23, and Glu 9 form apatch on the surface (FIG. 21), with Asp 7 centrally located in thepatch. Thus, it is reasonable to expect that these residues influenceeach other's ionization profile. In order to identify which of the threeresidues have a highly upshifted pK_(a), the H(C)CO spectrum of theprotein in 99% D₂O buffer at pH* 5.0 (direct pH meter reading) was thencollected. Asp 23 and Glu 9 showed larger deuterium isotope shifts (0.33and 0.32 ppm, respectively) than Asp 7 (0.18 ppm). These results showthat Asp 23 and Glu 9 are protonated to a greater degree than Asp 7.Thus, we concluded that Asp 23 and Glu 9 have highly upshifted pK_(a)'s,due to strong influence of Asp 7.

[0255] Mutational analysis

[0256] The spatial proximity of Asp 7 and 23, and Glu 9 explains theunfavorable electrostatic interactions in FNfn10 identified in thisstudy. At low pH where these residues are protonated and neutral, therepulsive interactions are expected to be mostly relieved. Thus, itshould be possible to improve the stability of FNfn10 at neutral pH, byremoving the electrostatic repulsion between these three residues.Because Asp 7 is centrally located among the three residues, it wasdecided to mutate Asp 7. Two mutants, D7N and D7K were prepared. Theformer neutralizes the negative charge with a residue of virtuallyidentical size. The latter places a positive charge at residue 7 andincreases the size of the side chain.

[0257] The ¹H, ¹⁵N-HSQC spectra of the two mutant proteins were nearlyidentical to that of the wild-type protein, indicating that thesemutations did not cause large structural perturbations (data not shown).The degrees of stability of the mutant proteins were then characterizedusing thermal and chemical denaturation measurements. Thermaldenaturation measurements were performed initially with 100 mM sodiumchloride, and 6.3 M urea was included to ensure reversible denaturationand to decrease the temperature of the thermal transition. All theproteins were predominantly folded in 6.3 M urea at room temperature.All the proteins underwent a cooperative transition, and the two mutantswere found to be significantly more stable than the wild type at neutralpH FIG. 22 and Table 10). Furthermore, these mutations almost eliminatedthe pH dependence of the conformational stability of FNfn10. Theseresults confirmed that destabilizing interactions involving Asp 7 inwild-type FNfn10 at neutral pH are the primary cause of the pHdependence. TABLE 10 The midpoint of thermal denaturation (in ° C.) ofwild-type and mutant FN3 in the presence of 6.3 M urea. pH 2.4 pH 7.0Protein 0.1 M NaCl 1 M NaCl 0.1 M NaCl 1 M NaCl wild type 72 82 62 70D7N 68 82 69 80 D7K 69 77 70 78

[0258] The effect of increased sodium chloride concentration on theconformational stability of the wild type and the two mutant proteinswas next investigated. All proteins were more stable in 1 M sodiumchloride than in 0.1 M sodium chloride (FIG. 22). The increase of thesodium chloride concentration elevated the T_(m) of the mutant proteinsby approximately 10° C. at both acidic and neutral pH (Table 10).Remarkably the wild-type protein was also equally stabilized at both pH,although it contains unfavorable interactions among the carboxyl groupsat neutral pH but not at acidic pH.

[0259] Chemical denaturation of FNfn10 proteins was monitored usingfluorescence emission from the single Trp residue of FNfn10 (FIG. 23).The free energies of unfolding at pH 6.0 and 4 M GuHCl were determinedto be 1.1 (±0.3), 1.7 (±0.2) and 1.4 (±0.1) kcal/mol for the wild type,D7N and D7K, respectively, indicating that the two mutations alsoincreased the conformational stability against chemical denaturation.

[0260] Determination of the pK_(a)'s of the side chain carboxyl groupsin the mutant proteins

[0261] The ionization properties of carboxyl groups in the two mutantproteins was investigated. The 2D H(C)CO spectra of the mutant proteinsat the high and low ends of the pH titration (pH ˜7 and ˜1.5,respectively) were nearly identical to the respective spectra of thewild type, except for the loss of the cross peaks for Asp 7 (data notshown). This similarity allowed for an unambiguous assignment ofresonances of the mutants, based on the assignments for wild-typeFNfn10. The pH titration experiments revealed that, except for Glu 9 andAsp 23, the behaviors of Asp and Glu carboxyl groups are very close totheir counterparts in the wild-type protein (FIG. 24 Panels A, C, D, Fand G, and Table 9), indicating that the two mutations have marginaleffects on the electrostatic environments for these carboxylates. Incontrast, the titration curves for E9 and D23 show significant changesupon mutation (FIG. 24 Panels B and E). The pK_(a) of D23 was lowered bymore than 1.6 and 1.4 pH units in the D7N and D7K mutants, respectively.These results clearly show that the repulsive interaction between D7 andD23 contributes to the increase in pK_(a) of Asp 23 in the wild-typeprotein, and that it was eliminated by the neutralization of thenegative charge at residue 7. The pK_(a) of Glu 9 was reduced by 0.4 pHunit by the D7N mutation, while it was decreased by 0.8 pH units in theD7K mutant. The greater reduction of Glu 9 pK_(a) by the D7K mutationsuggests that there is a favorable interaction between Lys 7 and Glu 9in this mutant protein.

[0262] Discussion

[0263] The present inventor has identified unfavorable electrostaticinteractions in FNfn10, and improved its conformational stability bymutations on the protein surface. The results demonstrate that repulsiveinteractions between like charges on protein surface significantlydestabilize a protein. The results are also consistent with recentreports by other groups (Loladze, V. V., Ibarra-Molero, B.,Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999) Biochemistry 38,16419-16423; Perl, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000)Nat Struct Biol 7, 380-383; Spector, S., Wang, M., Carp, S. A., Robblee,J., Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P. (2000)Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R.,Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L., Scholtz, J.M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849), in which proteinstability was improved by eliminating unfavorable electrostaticinteractions on the surface. In these studies, candidates for mutationswere identified by electrostatic calculations (Loladze, V. V.,Ibarra-Molero, B., Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999)Biochemistry 38, 16419-16423; Spector, S., Wang, M., Carp, S. A.,Robblee, J., Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P.(2000) Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L.R., Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L.,Scholtz, J. M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849) or bysequence comparison of homologous proteins with different stability(Perl, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000) Nat StructBiol 7, 380-383). The present strategy using pK_(a) determination usingNMR has both advantages and disadvantages over the other strategies. Thepresent method directly identifies residues that destabilize a protein.Also it does not depend on the availability of the high-resolutionstructure of the protein of interest. Electrostatic calculations mayhave large errors due to the flexibility of amino acid side chains onthe surface, and the uncertainty in the dielectric constant on theprotein surface and in the protein interior. For example, in the NMRstructure of FNfn10 (Main, A. L., Harvey, T. S., Baron, M., Boyd, J. &Campbell, I. D. (1992) Cell 71, 671-678), the root mean squareddeviations among 16 model structures for the O^(ε) atom of Glu residuesare 1.2-2.4 Å, and those for Lys N^(ζ) atoms are 1.5-3.1 Å. Suchuncertainties in atom position can potentially cause large differencesin calculation results. On the other hand, the present strategy requiresthe NMR assignments for carboxyl residues, and NMR measurements over awide pH range. Although recent advances in NMR spectroscopy have made itstraightforward to obtain resonance assignments for a small protein,some proteins may not be sufficiently soluble over the desired pH range.In addition, knowledge of the pK_(a) values of ionizable groups in thedenatured state is necessary for accurately evaluating contributions ofindividual residues to stability (Yang, A.-S. & Honig, B. (1992) Curr.Opin. Struct. Biol. 2, 40-45). Kuhlman et al. (Kuhlman, B., Luisi D. L.,Young, P. & Raleigh, D. P. (1999) Biochemistry 38, 4896-4903) showedthat pK_(a)'s of carboxylates in the denatured state has a considerablylarge range than those obtained from small model compounds. Despitethese limitations, the present method is applicable to many proteins.

[0264] The inventor showed that the unfavorable interactions involvingthe carboxyl groups of Asp 7, Glu 9 and Asp23 were no longer present ifthese groups are protonated at low pH or if Asp 7 was replaced with Asnor Lys. The similarity in the measured stability of the mutants and thewild type at low pH (Table 10) suggests that no other factorssignificantly contribute to the pH dependence of FNfn10 stability andthat the mutations caused minimal structural perturbations. The littlestructural perturbation was expected, since the carboxyl groups of thesethree residues are at least 50% exposed to the solvent, based on thesolvent accessible surface area calculation on the NMR structure (Main,A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I. D. (1992) Cell71, 671-678).

[0265] The difference in thermal stability of the wild-type proteinbetween acidic and neutral pH persisted in 1 M sodium chloride (Table10). Likewise, the wild-type protein exhibited a large pH-dependence instability in 4 M GuHCl (FIG. 18). Furthermore, upon the increase in thesodium chloride concentration from 0.1 to 1.0 M, the T_(m) of thewild-type and mutant proteins all increased by ˜10° C., which is in thesame magnitude as the change in T_(m) of the wild type by the pH shift.These data indicate that the unfavorable interactions identified in thisstudy were not effectively shielded in 1 M NaCl or in 4 M GuHCl. Becausethe effect of increased sodium chloride was uniform, this stabilizationeffect of sodium chloride is likely due to the nonspecific salting-outeffect (Timasheff, S. N. (1992) Curr. Op. Struct. Biol. 2, 35-39). Othergroups also reported little shielding effect of salts on electrostaticinteractions (Perutz, M. F., Gronenborn, A. M., Clore, G. M., Fogg, J.H. & Shih, D. T. (1985) J Mol Biol 183, 491-498; Hendsch, Z. S.,Jonsson, T., Sauer, R. T. & Tidor, B. (1996) Biochemistry 35,7621-7625). Electrostatic interactions are often thought to diminishwith increasing ionic strength, particularly if the site of interactionis highly exposed. Accordingly, the present data at neutral pH (Table10) showing no difference in the salt sensitivity between the wild typeand the mutants could be interpreted as Asp 7 not being responsible fordestabilizing electrostatic interactions. Although the reason for thissalt insensitivity is not yet clear, the present results provide acautionary note on concluding the presence and absence of electrostaticinteractions solely based on salt concentration dependence.

[0266] The carboxyl triad (Asp 7 and 23, and Glu 9) is highly conservedin FNfn10 from nine different organisms that were available in theprotein sequence databank at National Center for BiotechnologyInformation (www.ncbi.nlm.nih.gov). In these FNfn10 sequences, Asp 9 isconserved except one case where it is replaced with Asn, and Glu 9 iscompletely conserved. The position 23 is either Asp or Glu, preservingthe negative charge. As was discovered in this study, the interactionsamong these residues are destabilizing. Thus, their high conservation,despite their negative effects on stability, suggests that theseresidues have functional importance in the biology of fibronectin. Inthe structure of a four-FN3 segment of human fibronectin (Leahy, D. J.,Aukhil, I. & Erickson, H. P. (1996) Cell 84, 155-164), these residuesare not directly involved in interactions with adjacent domains. Alsothese residues are located on the opposite face of FNfn10 from theintegrin-binding RGD sequence in the FG loop (FIG. 21). Therefore, it isnot clear why these destabilizing residues are almost completelyconserved in FNfn10. In contrast, no other FN3 domains in humanfibronectin contain this carboxyl triad (for a sequence alignment, seeref Main, A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I. D.(1992) Cell 71, 671-678). The carboxyl triad of FNfn10 may be involvedin important interactions that have not been identified to date.

[0267] Clarke et al. (Clarke, J., Hamill, S. J. & Johnson, C. M. (1997)J Mol Biol 270, 771-778) reported that the stability of the third FN3 ofhuman tenascin (TNfn3) increases as pH was decreased from 7 to 5.Although they could not perform stability measurements below pH 5 due toprotein aggregation, the pH dependence of TNfn3 resembles that of FNfn10shown in FIG. 18. TNfn3 does not contain the carboxylate triad atpositions 7, 9 and 23 (Leahy, D. J., Hendrickson, W. A., Aukhil, I. &Erickson, H. P. (1992) Science 258, 987-991), indicating that thedestabilization of TNfn3 at neutral pH is caused by a differentmechanism from that for FNfn10. A visual inspection of the TNfn3structure revealed that it has a large number of carboxyl groups, andthat Glu 834 and Asp 850 (numbering according to ref Leahy, D. J.,Hendrickson, W. A., Aukhil, I. & Erickson, H. P. (1992) Science 258,987-991) forms a cross-strand pair. It will be interesting to examinewhether altering this pair can increase the stability of TNfn3.

[0268] In conclusion, a strategy has been described to experimentallyidentify unfavorable electrostatic interactions on the protein surfaceand improve the protein stability by relieving such interactions. Thepresent results have demonstrated that forming a repulsive interactionbetween carboxyl groups significantly destabilize a protein. This is incontrast to the small contributions of forming a solvent-exposed ionpair. Unfavorable electrostatic interactions on the surface seem quitecommon in natural proteins. Therefore, optimization of the surfaceelectrostatic properties provides a generally applicable strategy forincreasing protein stability (Loladze, V. V., Ibarra-Molero, B.,Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999) Biochemistry 38,16419-16423; Perl, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000)Nat Struct Biol 7, 380-383; Spector, S., Wang, M., Carp, S. A., Robblee,J., Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P. (2000)Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R.,Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L., Scholtz, J.M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849). In addition,repulsive interactions between carboxylates can be exploited fordestabilizing undesirable, alternate conformations in protein design(“negative design”).

EXAMPLE XX An Extension of the Carboxyl-terminus of the MonobodyScaffold

[0269] The wild-type protein used for stability measurements isdescribed under Example 19. The carboxyl-terminus of the monobodyscaffold was extended by four amino acid residues, namely, amino acidresidues (Glu-Ile-Asp-Lys) (SEQ ID NO:119), which are the ones thatimmediately follow FNfn10 of human fibronectin. The extension wasintroduced into the FNfn10 gene using standard PCR methods. Stabilitymeasurements were performed as described under Example 19. The freeenergy of unfolding of the extended protein was 7.4 kcal mol⁻¹ at pH 6.0and 30° C., very close to that of the wild-type protein (7.7 kcalmol⁻¹). These results demonstrate that the C-terminus of the monobodyscaffold can be extended without decreasing its stability.

[0270] The complete disclosure of all patents, patent documents andpublications cited herein are incorporated by reference as ifindividually incorporated. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

References

[0271] Alzari, P. N., Lascombe, M.-B. & Poljak, R. J. (1988)Three-dimensional structure of antibodies. Annu. Rev. Immunol. 6,555-580.

[0272] Archer, S. J., Ikura, M., Torchia, D. A. & Bax, A. (1991) Analternative 3D NMR technique for correlating backbone 15N with sidechain Hb resonances in large proteins J. Magn. Reson. 95, 636-641.

[0273] Aukhil, I., Joshi, P., Yan, Y. & Erickson, H. P. (1993) Cell- andheparin-binding domains of the hexabrachion arm identified by tenascinexpression protein J. Biol. Chem. 268, 2542-2553.

[0274] Barbas, C. F., III, Kang, A. S., Lerner, R. A. & Benkovic, S. J.(1991) Assembly of combinatorial antibody libraries on phage surfaces:the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978-7982.

[0275] Barbas, C. F., III, Bain, J. D., Hoekstra, D. M. & Lerner, R. A.(1992) Semisynthetic combinatorial libraries: A chemical solution to thediversity problem Proc. Natl. Acad. Sci. USA 89,4457-4461.

[0276] Baron, M., Main, A. L., Driscoll, P. C., Mardon, H. J., Boyd, J.& Campbell, I. D. (1992) ¹H NMR assignment and secondary structure ofthe cell adhesion type II module of fibronectin Biochemistry 31,2068-2073.

[0277] Baron, M., Norman, D. G. & Campbell, I. D. (1991) Protein modulesTrends Biochem. Sci. 16, 13-17.

[0278] Bass, S., Greene, R. & Wells, J. A. (1990) Hormone phage: Anenrichment method for variant proteins with altered binding propertiesProteins: Struct. Funct. Genet. 8, 309-314.

[0279] Bax, A. & Grzesiek, S. (1993) Methodological advances in proteinNMR. Acc. Chem. Res. 26, 131-138.

[0280] Becktel, W. J. & Schellman, J. A. (1987) Protein stabilitycurves. Biopolymer 26, 1859-1877.

[0281] Bhat, T. N., Bentley, G. A., Boulot, G., Greene, M. I., Tello,D., Dall'acqua, W., Souchon, H., Schwarz, F. P., Mariuzza, R. A. &Poljak, R. J. (1994) Bound water molecules and conformationalstabilization help mediate an antigen-antibody association. Proc. Natl.Acad. Sci. USA 91, 1089-1093.

[0282] Bianchi, E., Venturini, S., Pessi, A., Tramontano, A. & Sollazzo,M. (1994) High level expression and rational mutagenesis of a designedprotein, the minibody. From an insoluble to a soluble molecule. J. Mol.Biol. 236, 649.659.

[0283] Billeter, M., Neri, D., Otting, G., Qian, Y. Q. & Wüthrich, K.(1992) Precise vicinal coupling constants 3JHNa in proteins fromnonlinear fits of J-modulated [¹⁵N, ¹H]-COSY experiments. J. Biomol. NMR2, 257-274.

[0284] Bodenhausen, G. & Ruben, D. J. (1980) Natural abundancenitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys.Lett. 69, 185-189.

[0285] Bork, P. & Doolittle, R. F., PNAS 89:8990-8994 (1992).

[0286] Bork, P. & Doolittle, R. F. (1992) Proposed acquisition of ananimal protein domain by bacteria. Proc. Natl. Acad. Sci. USA 89,8990-8994.

[0287] Bork, P., Hom, L. & Sander, C. (1994) The immunoglobulin fold.Structural classification, sequence patterns and common core. J. Mol.Biol. 242, 309-320.

[0288] Brünger, A. T. (1992) X-PLOR (Version 3.1): A system for X-raycrystallography and NMR., Yale Univ. Press, New Haven.

[0289] Burke, T., Bolger, R., Checovich, W. & Lowery, R. (1996) in Phagedisplay of peptides and proteins (Kay, B. K., Winter, J. and McCafferty,J., Ed.) Vol. pp 305-326, Academic Press, San Diego. Campbell, I. D. &Spitzfaden, C. (1994) Building proteins with fibronectin type IIImodules Structure 2, 233-337.

[0290] Chen, Y., Reizer, J., Saier, M. H., Fairbrother, W. J. & Wright,P. E. (1993) Mapping the binding interfaces of the proteins of thebacterial phaphotransferase system, HPr and IIAglc. Biochemistry 32,32-37.

[0291] Clarke, J., Hamill, S. J. & Johnson, C. M. (1997) J Mol Biol 270,771-778.

[0292] Clackson & Wells, (1994) Trends Biotechnology 12, 173-184.

[0293] Clore, G. M. & Gronenborn, A. M. (1991) Structure of largerproteins in solution: Three- and four-dimensional heteronuclear NMRspectroscopy. Science 252, 1390-1399.

[0294] Corey, D. R., Shiau, A. K., Q., Y., Janowski, B. A. & Craik, C.S. (1993) Trypsin display on the surface of bacteriophage. Gene 128,129-134.

[0295] Cota, E. & Clarke, J. (2000) Protein Sci 9, 112-120.

[0296] Creighton, T. E. (1993) Proteins: structures and molecularproperties, Freeman, N.Y.

[0297] Dao-pin, S., Sauer, U., Nicholson, H. & Matthews, B. W. (1991)Biochemistry 30, 7142-7153.

[0298] Davies, J. & Riechmann, L. (1996). Single antibody domains assmall recognition units: design and in vitro antigen selection ofcamelized, human VH domains with improved protein stability. ProteinEng., 9(6), 531-537.

[0299] Davies, J. & Riechmann, L. (1995) Antobody VH domains as smallrecognition units. Bio/Technol. 13, 475-479.

[0300] Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J.& Bax, A. (1995) NMRPipe: a multidimensional spectral processing systembased on UNIX pipes. J. Biomol. NMR 6, 277-293.

[0301] Deng, W. P. & Nickoloff, J. A. (1992) Site-directed mutagenesisof virtually any plasmid by eliminating a unique site. Anal. Biochem.200, 81-88.

[0302] deVos, A. M., Ultsch, M. & Kossiakoff, A. A. (1992) Human Growthhormone and extracellular domain of its receptor: crystal structure ofthe complex. Science 255, 306-312.

[0303] Dickinson, C. D., Veerapandian, B., Dai, X. P., Hamlin, R. C.,Xuong, N.-H., Ruoslahti, E. & Ely, K. R. (1994) Crystal structure of thetenth type III cell adhesion module of human fibronectin J. Mol. Biol.236, 1079-1092.

[0304] Dill, K. A. (1990) Biochemistry 29, 7133-7155.

[0305] Djavadi-Ohaniance, L., Goldberg, M. E. & Friguet, B. (1996) inAntibody Engineering. A Practical Approach (McCafferty, J., Hoogenboom,H. R. and Chiswell, D. J., Ed.) Vol. pp. 77-97, Oxford Univ. Press,Oxford.

[0306] Dougall, W. C., Peterson, N. C. & Greene, M. I. (1994)Antibody-structure-based design of pharmacological agents. TrendsBiotechnol. 12, 372-379.

[0307] Garrett, D. S., Powers, R., Gronenborn, A. M. & Clore, G. M.(1991) A common sense approach to peak picking in two-, three- andfour-dimensional spectra using automatic computer analysis of contourdiagrams. J. Magn. Reson. 95, 214-220.

[0308] Ghosh, G., Van Duyne, G., Ghosh, S. & Sigler, P. B. (1995)Structure of NF-kB p50 homodimer bound to a kB site. Nature 373,303-310.

[0309] Gribskov, M., Devereux, J. & Burgess, R. R. (1984) The codonpreference plot: graphic analysis of protein coding sequences andprediction of gene expression. Nuc. Acids. Res. 12, 539-549.

[0310] Grimsley, G. R., Shaw, K. L., Fee, L. R., Alston, R. W.,Huyghues-Despointes, B. M., Thurlkill, R. L., Scholtz, J. M. & Pace, C.N. (1999) Protein Sci 8, 1843-1849.

[0311] Groneborn, A. M., Filpula, D. R., Essig, N. Z., Achari, A.,Whitlow, M., Wingfield, P. T. & Clore, G. M. (1991) A novel, highlystable fold of the immunoglobulin binding domain of Streptococcalprotein G. Science 253, 657-661.

[0312] Gronenborn, A. M. & Clore, G. M. (1993) Identification of thecontact surface of a Streptococcal protein G domain complexed with ahuman Fc fragment. J. Mol. Biol 233, 331-335.

[0313] Grzesiek, S., Anglister, J. & Bax, A. (1993) Correlation ofbackbone amide and aliphatic side-chain resonances in 13C/15N-enrichedproteins by isotropic mixing of 13C magnetization. J. Magn. Reson. B101, 114-119.

[0314] Grzesiek, S. & Bax, A. (1992) Correlating backbone amide and sidechain resonances in larger proteins by multiple relayed triple resonanceNMR. J. Am. Chem. Soc. 114, 6291-6293.

[0315] Grzesiek, S. & Bax, A. (1993) Amino acid type determination inthe sequential assignment procedure of uniformly 13C/15N-enrichedproteins. J. Biomol. NMR 3, 185-204.

[0316] Harlow, E. & Lane, D. (1988) Antibodies. A laboratory manual,Cold Spring Harbor Laboratory, Cold Spring Harbor.

[0317] Harpez, Y. & Chothia, C. (1994) Many of the immunoglobulinsuperfamily domains in cell adhesion molecules and surface receptorsbelong to a new structural set which is close to that containingvariable domains J. Mol. Biol. 238, 528-539.

[0318] Hawkins, R. E., Russell, S. J., Bay, M. & Winter, G. (1193) Thecontribution of contact and non-contact residues of antibody in theaffinity of binding to antigen. The interaction of mutant D1.3antibodies with lysozyme. J. Mol. Biol. 234, 958-964.

[0319] Hendsch, Z. S., Jonsson, T., Sauer, R. T. & Tidor, B. (1996)Biochemistry 35, 7621-7625.

[0320] Hendsch, Z. S. & Tidor, B. (1994) Protein Sci. 3, 211-226.

[0321] Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. 90, 6444-6448.

[0322] Hu, S-z., et al., Cancer Res. 56:3055-3061 (1996).

[0323] Ikura, M. & Bax, A. (1992) Isotope-filtered 2D NMR of aprotein-peptide complex: study of a skeletal muscle myosin light chainkinase fragment bound to calmodulin. J. Am. Chem. Soc. 114, 2433-2440.

[0324] Ikura, M., Kay, L. E. & Bax, A. (1991) Improved three-dimensional1H-13C-1H correlation spectroscopy of a 13C-labeled protein usingconstant-time evolution. J. Biomol. NMR 1, 299-304.

[0325] Jacobs, J. & Schultz, P. G. (1987) Catalytic antibodies. J. Am.Chem. Soc. 109, 2174-2176.

[0326] Johnson, B. A. & Blevins, R. A. (1994) J. Biomol. NMR 4, 603-614.

[0327] Janda, K. D., et al., Science 275:945-948 (1997).

[0328] Jones, E. Y. (1993) The immunoglobulin superfamily Curr. Opinionstruct. Biol. 3, 846-852.

[0329] Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S. & Winter,G. (1986) Replacing the complementarity-determining regions in a humanantibody with those from a mouse Nature 321, 522-525.

[0330] Kabsch, W. & Sander, C. (1983) Dictionary of protein secondarystructure: pattern recognition of hydrogen-bonded and geometricalfeatures. Biopolymers 22,2577-2637.

[0331] Kamtekar, S. Schiffer J M, Xiong H, Babik J M, Hecht M H. (1993)Protein design by binary patterning of polar and nonpolar amino acids.Science 262(5140):1680-1685.

[0332] Kauzmann, W. (1959) Adv. Prot. Chem. 14, 1-63.

[0333] Kay, L. E. (1995) Field gradient techniques in NMR spectroscopy.Curr. Opinion Struct. Biol. 5, 674-681.

[0334] Kay, L. E., Keifer, P. & Saarinen, T. (1992) Pure absorptiongradient enhanced heteronuclear single quantum correlation spectroscopywith improved sensitivity J. Am. Chem. Soc. 114, 10663-10665.

[0335] Kay, L. E. (1993) J. Am. Chem. Soc. 115, 2055-2057.

[0336] Kay, L. E., Xu, G.-Y. & Singer, A. U. (1993) A Gradient-EnhancedHCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlationsin H2O Samples of Proteins. J. Magn. Reson. B 101, 333-337.

[0337] Koide, S., Bu, Z., Risal, D., Pham, T.-N., Nakagawa, T., Tamura,A. & Engelman, D. M. (1999) Biochemistry 38, 4757-4767.

[0338] Koide, A., Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol.Biol. 284, 1141-1151.

[0339] Koide, S., Dyson, H. J. & Wright, P. E. (1993) Characterizationof a folding intermediate of apoplastcyanin trapped by prolineisomerization. Biochemistry 32, 12299-12310.

[0340] Kornblihtt, A. R., Umezawa, K., Vibe-Pederson, K. & Baralle, F.E. (1985) Primary structure of human fibronectin: differential splicingmay generate at least 10 polypeptides from a single gene EMBO J. 4,1755-1759.

[0341] Kraulis, P. (1991) MOLSCRIPT: a program to produce both detailedand scnematic plots of protein structures. J. Appl. Cryst. 24, 946-950.

[0342] Kuhlman, B., Luisi, D. L., Young, P. & Raleigh, D. P. (1999)Biochemistry 38, 4896-4903.

[0343] Kunkel, T. A. (1985) Rapid and efficient site-specificmutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82,488-492.

[0344] Leahy, D. J., Aukhil, I. & Erickson, H. P. (1996) Cell 84,155-164.

[0345] Leahy, D. J., Hendrickson, W. A., Aukhil, I. & Erickson, H. P.(1992) Structure of a fibronectin type III domain from tenascin phasedby MAD analysis of the selenomethionlyl protein Science 258, 987-991.

[0346] Lee, W., Revington, M. J., Arrowsmith, C. & Kay, L. E. (1994) Apulsed field gradient isotope-filtered 3D1.3C HMQC-NOESY experiment forextracting intermolecular NOE contacts in molecular complexes. FEBSlett. 350, 87-90.

[0347] Lerner, R. A. & Barbas III, C. F., Acta Chemica Scandinavica, 50672-678 (1996).

[0348] Lesk, A. M. & Tramontano, A. (1992) Antibody structure andstructural predictions useful in guiding antibody engineering. InAntibody engineering. A practical guide. (Borrebaeck, C. A. K., Ed.)Vol. W. H. Freeman & Co., New York.

[0349] Li, B., Tom, J. Y., Oare, D., Yen, R., Fairbrother, W. J., Wells,J. A. & Cunningham, B. C. (1995) Minimization of a polypeptide hormoneScience 270, 1657-1660.

[0350] Litvinovich, S. V., Novokhatny, V. V., Brew, S. A. & Inhgam, K.C. (1992) Reversible unfolding of an isolated heparin and DNA bindingfragment, the first type III module from fibronectin. Biochim. Biophys.Acta 1119, 57-62.

[0351] Logan, T. M., Olejniczak, E. T., Xu, R. X. & Fesik, S. W. (1992)Side chain and backbone assignments in isotopically labeled proteinsfrom two heteronuclear triple resonance experiments. FEBS lett. 314,413-418.

[0352] Loladze, V. V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. &Makhatadze, G. I. (1999) Biochemistry 38, 16419-16423.

[0353] Main, A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I. D.(1992) The three-dimensional structure of the tenth type III module offibronectin: an insight into RGD-mediated interactions. Cell 71,671-678.

[0354] Malakauskas, S. M. & Mayo, S. L. (1998) Nat Struct Biol 5,470-475.

[0355] Masat, L., et al., (1994) PNAS 91:893-896.

[0356] Martin, F., Toniatti, C., Ciliberto, G., Cortese, R. & Sollazzo,M. (1994) The affinity-selection of a minibody polypeptide inhibitor ofhuman interleukin-6. EMBO J 13, 5303-5309.

[0357] Martin, M. T., Drug Discov. Today, 1:239-247 (1996).

[0358] McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J.(1990) Phage antibodies: filamentous phage displaying antibody variabledomains. Nature 348, 552-554.

[0359] McConnell, S. J., & Hoess, R. H., J. Mol. Biol. 250:460-470(1995).

[0360] McIntosh, L. P., Hand, G., Johnson, P. E., Joshi, M. D., Koerner,M., Plesniak, L. A., Ziser, L., Wakarchuk, W. W. & Withers, S. G. (1996)Biochemistry 35, 9958-9966.

[0361] Metzler, W. J., Leiting, B., Pryor, K., Mueller, L. & Farmer, B.T. I. (1996) The three-dimensional solution structure of the SH2 domainfrom p55blk kinase. Biochemistry 35, 6201-6211.

[0362] Minor, D. L. J. & Kim, P. S. (1994) Measurement of theβ-sheet-forming propensities of amino acids. Nature 367, 660-663.

[0363] Muhandiram, D. R., Xu, G. Y. & Kay, L. E. (1993) Anenhanced-sensitivity pure absorption gradient 4D 15N, 13C-edited NOESYexperiment. J. Biomol. NMR 3, 463-470.

[0364] Müller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L. &Harrison, S. C. (1995) Structure of the NH-kB p50 homodimer bound toDNA. Nature 373, 311-117.

[0365] Myers, J. K., Pace, C. N. & Scholtz, J. M. (1995) Protein Sci. 4,2138-2148.

[0366] Nilges, M., Clore, G. M. & Gronenborn, A. M. (1988) Determinationof three-dimensional structures of proteins from interproton distancedata by hybrid distance geometry-dynamical simulated annealingcalculations. FEBS lett. 229, 317-324.

[0367] Nilges, M., Kuszewski, J. & Brünger, A. T. (1991) inComputational aspects of the study of biological macromolecules bynuclear magnetic resonance spectroscopy. (Hoch, J. C., Poulsen, F. M.and Redfield, C., Ed.) Vol. pp. 451-455, Plenum Press, New York.

[0368] O'Neil et al., (1994) in Techniques in Protein Chemistry V(Crabb, L., ed.) pp. 517-524, Academic Press, San Diego.

[0369] O'Neil, K. T. & Hoess, R. H. (1995) Phage display: proteinengineering by directed evolution. Curr. Opinion Struct. Biol. 5,443-449.

[0370] Pace, C. N. & Scholtz, J. M. (1997) Measuring the conformationalstability of a protein. In Protein structure. A practical approach(Creighton, T. E., Ed.) Vol. pp. 299-321, IRL Press, Oxford.

[0371] Pace, C. N., Shirley, B. A., McNutt, M. & Gajiwala, K. (1996)Faseb J 10, 75-83.

[0372] Pace, C. N., Laurents, D. V. & Erickson, R. E. (1992)Biochemistry 31, 2728-2734.

[0373] Parmley, S. F. & Smith, G. P. (1988) Antibody-selectablefilamentous fd phage vectors: affinity purification of target genes Gene73, 305-318.

[0374] Pascal, S. M., Muhandiram, D. R., Yamazaki, T., Forman-Kay, J. D.& Kay, L. E. (1994a) Simultaneous acquisition of 15N- and 13C-edited NOEspectra of proteins dissolved in H2O. J. Magn. Reson. B 103, 197-201.

[0375] Pascal, S. M., Singer, A. U., Gish, G., Yamazaki, T., Shoelson,S. E., Pawson, T., Kay, L. E. & Fornan-Kay, J. D. (1994b) Nuclearmagnetic resonance structure of an SH2 domain of phospholipase C-glcomplexed with a high affinity binding peptide. Cell 77, 461-472.

[0376] Perl, D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000) NatStruct Biol 7, 380-383.

[0377] Perutz, M. F., Gronenborn, A. M., Clore, G. M., Fogg, J. H. &Shih, D. T. (1985) J Mol Biol 183, 491-498.

[0378] Pessi, A., Bianchi, E., Crameri, A., Venturini, S., Tramontano,A. & Sollazzo, M. (1993) A designed metal-binding protein with a novelfold. Nature 362, 3678-369.

[0379] Pierschbacher, M. D. & Ruoslahti, E. (1984) Nature 309, 30-33.

[0380] Plaxco, K. W., Spitzfaden, C., Campbell, I. D. & Dobson, C. M.(1996) Proc. Natl. Acad. Sci. USA 93, 10703-10706.

[0381] Plaxco, K. W., Spitzfaden, C., Campbell, 1. D. & Dobson, C. M.(1997) J. Mol. Biol. 270, 763-770.

[0382] Rees, A. R., Staunton, D., Webster, D. M., Searle, S. J., Henry,A. H. & Pedersen, J. T. (1994) Antibody design: beyond the naturallimits. Trends Biotechnol. 12, 199-206.

[0383] Roberts et al., (1992) Proc. Natl. Acad. Sci. USA 89, 2429-2433.

[0384] Rosenblum, J. S. & Barbas, C. F. I. (1995) in AntobodyEngineering (Borrenbaeck, C. A. K., Ed.) Vol. pp 89-116, OxfordUniversity Press, Oxford.

[0385] Sali, D., Bycroft, M. & Fersht, A. R. (1991) J. Mol. Biol. 220,779-788.

[0386] Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) MolecularCloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor.

[0387] Sandhu, G. S., Aleff, R. A. & Kline, B. C. (1992) Dual asymmetricPCR: one-step construction of synthetic genes. BioTech. 12, 14-16.

[0388] Santoro, M. M. & Bolen, D. W. (1988) Unfolding free energychanges determined by the linear extrapolation method. 1. Unfolding ofphenylmethanesulfonyl a-chymotrypsin using different denaturantsBiochemistry 27, 8063-8068.

[0389] Smith, G. P. & Scott, J. K. (1993) Libraries of peptides andproteins displayed on filamentous phage. Methods Enzymol. 217, 228-257.

[0390] Smith, G. P. (1985) Filamentous fusion phage: novel expressionvectors that display cloned antigens on the virion surface. Science 228,1315-1317.

[0391] Smith, C. K., Munson, M. & Regan, L. (1995). Studying α-helix andβ-sheet formation in small proteins. Techniques Prot. Chem., 6, 323-332.

[0392] Smith, C. K., Withka, J. M. & Regan, L. (1994) A thermodynamicscale for the b-sheet forming tendencies of the amino acids.Biochemistry 33, 5510-5517.

[0393] Smyth, M. L. & von Itzstein, M. (1994) Design and synthesis of abiologically active antibody mimic based on an antibody-antigen crystalstructure. J. Am. Chem. Soc. 116, 2725-2733.

[0394] Spector, S., Wang, M., Carp, S. A., Robblee, J., Hendsch, Z. S.,Fairman, R., Tidor, B. & Raleigh, D. P. (2000) Biochemistry 39, 872-879.

[0395] Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W.(1990) Use of T7 RNA polymerase to direct expression of cloned genesMethods Enzymol. 185, 60-89.

[0396] Suzuki, H. (1994) Recent advances in abzyme studies. J. Biochem.115, 623-628.

[0397] Tello, D., Goldbaum, F. A., Mariuzza, R. A., Ysern, X., Schwarz,F. P. & Poljak, R. J. (1993) Immunoglobulin superfamily interactions.Biochem. Soc. Trans. 21, 943-946.

[0398] Thomas, N. R. (1994) Hapten design for the generation ofcatalytic antibodies. Appl. Biochem. Biotech. 47, 345-372.

[0399] Timasheff, S. N. (1992) Curr. Op. Struct. Biol. 2, 35-39.

[0400] Verhoeyen, M., Milstein, C. & Winter, G. (1988) Reshaping humanantibodies: Grafting an antilysozyme activity. Science 239, 1534-1536.

[0401] Venturini et al., (1994) Protein Peptide Letters 1, 70-75.

[0402] Vuister, G. W. & Bax, A. (1992) Resolution enhancement andspectral editing of uniformly 13C-enriched proteins by homonuclearbroadband 13C decoupling. J. Magn. Reson. 98, 428-435.

[0403] Vuister, G. W., Clore, G. M., Gronenborn, A. M., Powers, R.,Garrett, D. S., Tschudin, R. & Bax, A. (1993) Increased resolution andimproved spectral quality in four-dimensional 13C/13C-separatedHMQC-NOESY-HMQC spectra using pulsed filed gradients. J. Magn. Reson. B101, 210-213.

[0404] Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T. & Winter,G. (1989) Binding activities of a repertoire of single immunoglobulinvariable domains secreted from Escherichia coli Nature 341, 554-546.

[0405] Webster, D. M., Henry, A. H. & Rees, A. R. (1994)Antibody-antigen interactions Curr. Opinion Struct. Biol. 4, 123-129.

[0406] Williams, A. F. & Barclay, A. N., Ann. Rev. Immunol. 6:381-405(1988).

[0407] Wilson, I. A. & Stanfield, R. L. (1993) Antibody-antigeninteractions. Curr. Opinion Struct. Biol. 3, 113-118.

[0408] Wilson, I. A. & Stanfield, R. L. (1994) Antibody-antigeninteractions: new structures and new conformational changes Curr.Opinion Struct Biol. 4, 857-867.

[0409] Winter, G., Griffiths, A. D., Hawkins, R. E. & Hoogenboom, H. R.(1994) Making antibodies by phage display technology Annu. Rev. Immunol.12, 433-455.

[0410] Wiseman, T., Williston, S., Brandts, J. F. & Lin, L.-N. (1989)Rapid measurement of binding constants and heats of binding using a newtitration calorimeter. Anal. Biolchem. 179, 131-137.

[0411] Wittenkind, M. & Mueller, L. (1993) HNCACB, a high-sensitivity 3DNMR experiment to correlate amide-proton and nitrogen resonances withthe alpha- and beba-carbon resonances in proteins J. Magn. Reson. B 101,201-205.

[0412] Wu, T. T., Johnson, G. & Kabat, E. A. (1993) Length distributionof CDRH3 in antibodies Proteins: Struct. Funct. Genet. 16, 1-7.

[0413] Wüthrich, K. (1986) NMR of proteins and nucleic acids, John Wiley& Sons, New York.

[0414] Yamazaki, T., Fornan-Kay, J. D. & Kay, L. E. (1993)Two-Dimensional NMR Experiments for Correlating 13C-beta and1H-delta/epsilon Chemical Shifts of Aromatic Residues in 13C-LabeledProteins via Scalar Couplings. J. Am. Chem. Soc. 115, 11054.

[0415] Yang, A.-S. & Honig, B. (1992) Curr. Opin. Struct. Biol. 2,40-45.

[0416] Zhang, O., Kay, L. E., Olivier, J. P. & Forman-Kay, J. D. (1994)Backbone 1H and 15N resonance assignments of the N-terminal SH3 domainof drk in folded and unfolded states using enhanced-sensitivity pulsedfield gradient NMR techniques. J. Biomol. NMR 4, 845-858.

1 121 1 14 PRT Unknown Anti-hen egg lysozyme (HEL) antibody. 1 Ala ArgGlu Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gly 1 5 10 2 17 PRT UnknownAn anti-HEL single VH domain termed VH8. 2 Ala Arg Gly Ala Val Val SerTyr Tyr Ala Met Asp Tyr Trp Gly Gln 1 5 10 15 Gly 3 16 PRT Homo sapiens3 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile 1 5 1015 4 12 PRT Artificial Sequence Mutant D1.3-1. 4 Tyr Ala Glu Arg Asp TyrArg Leu Asp Tyr Pro Ile 1 5 10 5 12 PRT Artificial Sequence MutantD1.3-2. 5 Tyr Ala Val Arg Asp Tyr Arg Leu Asp Tyr Pro Ile 1 5 10 6 16PRT Artificial Sequence Mutant D1.3-3. 6 Tyr Ala Val Arg Asp Tyr Arg LeuAsp Tyr Ala Ser Ser Lys Pro Ile 1 5 10 15 7 13 PRT Artificial SequenceMutant D1.3-4. 7 Tyr Ala Val Arg Asp Tyr Arg Leu Asp Tyr Lys Pro Ile 1 510 8 11 PRT Artificial Sequence Mutant D1.3-5. 8 Tyr Ala Val Arg Asp TyrArg Ser Lys Pro Ile 1 5 10 9 14 PRT Artificial Sequence Mutant D1.3-6. 9Tyr Ala Val Thr Arg Asp Tyr Arg Leu Ser Ser Lys Pro Ile 1 5 10 10 15 PRTArtificial Sequence Mutant D1.3-7. 10 Tyr Ala Val Thr Glu Arg Asp TyrArg Leu Ser Ser Lys Pro Ile 1 5 10 15 11 15 PRT Artificial SequenceMutant VH8-1. 11 Tyr Ala Val Ala Val Val Ser Tyr Tyr Ala Met Asp Tyr ProIle 1 5 10 15 12 16 PRT Artificial Sequence Mutant VH8-2. 12 Tyr Ala ValThr Ala Val Val Ser Tyr Tyr Ala Ser Ser Lys Pro Ile 1 5 10 15 13 59 DNAArtificial Sequence Oligonucleotide FN1F. 13 cgggatccca tatgcaggtttctgatgttc cgcgtgacct ggaagttgtt gctgcgacc 59 14 55 DNA ArtificialSequence Oligonucleotide FN1R. 14 taactgcagg agcatcccag ctgatcagcaggctagtcgg ggtcgcagca acaac 55 15 51 DNA Artificial SequenceOligonucleotide FN2F. 15 ctcctgcagt taccgtgcgt tattaccgta tcacgtacggtgaaaccggt g 51 16 39 DNA Artificial Sequence Oligonucleotide FN2R. 16gtgaattcct gaaccgggga gttaccaccg gtttcaccg 39 17 46 DNA ArtificialSequence Oligonucleotide FN3F. 17 aggaattcac tgtacctggt tccaagtctactgctaccat cagcgg 46 18 38 DNA Artificial Sequence Oligonucleotide FN3R.18 gtatagtcga cacccggttt caggccgctg atggtagc 38 19 32 DNA ArtificialSequence Oligonucleotide FN4F. 19 cgggtgtcga ctataccatc actgtatacg ct 3220 55 DNA Artificial Sequence Oligonucleotide FN4R. 20 cgggatccgagctcgctggg ctgtcaccac ggccagtaac agcgtataca gtgat 55 21 35 DNAArtificial Sequence Oligonucleotide FN5F. 21 cagcgagctc caagccaatctcgattaact accgt 35 22 37 DNA Artificial Sequence Oligonucleotide FN5R.22 cgggatcctc gagttactag gtacggtagt taatcga 37 23 38 DNA ArtificialSequence Oligonucleotide FN5R′. 23 cgggatccac gcgtgccacc ggtacggtagttaatcga 38 24 44 DNA Artificial Sequence Oligonucleotide gene3F. 24cgggatccac gcgtccattc gtttgtgaat atcaaggcca atcg 44 25 39 DNA ArtificialSequence Oligonucleotide gene3R. 25 ccggaagctt taagactcct tattacgcagtatgttagc 39 26 36 DNA Artificial Sequence Oligonucleotide 38TAABg1II.26 ctgttactgg ccgtgagatc taaccagcga gctcca 36 27 51 DNA ArtificialSequence Oligonucleotide BC3. 27 gatcagctgg gatgctcctn nknnknnknnknnktattac cgtatcacgt a 51 28 57 DNA Artificial Sequence OligonucleotideFG2. 28 tgtatacgct gttactggcn nknnknnknn knnknnknnk tccaagccaa tctcgat57 29 47 DNA Artificial Sequence Oligonucleotide FG3. 29 ctgtatacgctgttactggc nnknnknnkn nkccagcgag ctccaag 47 30 51 DNA ArtificialSequence Oligonucleotide FG4. 30 catcactgta tacgctgtta ctnnknnknnknnknnktcc aagccaatct c 51 31 5 PRT Artificial Sequence The sequence ofthe BC loop of ubiquitin- binding monobody clone 211. 31 Cys Ala Arg ArgAla 1 5 32 7 PRT Artificial Sequence The sequence of the FG loop ofubiquitin- binding monobody clone 211. 32 Arg Trp Ile Pro Leu Ala Lys 15 33 5 PRT Artificial Sequence The sequence of the BC loop of ubiquitin-binding monobody clone 212. 33 Cys Trp Arg Arg Ala 1 5 34 7 PRTArtificial Sequence The sequence of the FG loop of ubiquitin- bindingmonobody clone 212. 34 Arg Trp Val Gly Leu Ala Trp 1 5 35 5 PRTArtificial Sequence The sequence of the BC loop of ubiquitin- bindingmonobody clone 213. 35 Cys Lys His Arg Arg 1 5 36 7 PRT ArtificialSequence The sequence of the FG loop of ubiquitin- binding monobodyclone 213. 36 Phe Ala Asp Leu Trp Trp Arg 1 5 37 5 PRT ArtificialSequence The sequence of the BC loop of ubiquitin- binding monobodyclone 214. 37 Cys Arg Arg Gly Arg 1 5 38 7 PRT Artificial Sequence Thesequence of the FG loop of ubiquitin- binding monobody clone 214. 38 ArgGly Phe Met Trp Leu Ser 1 5 39 5 PRT Artificial Sequence The sequence ofthe BC loop of ubiquitin- binding monobody clone 215. 39 Cys Asn Trp ArgArg 1 5 40 7 PRT Artificial Sequence The sequence of the FG loop ofubiquitin- binding monobody clone 215. 40 Arg Ala Tyr Arg Tyr Arg Trp 15 41 5 PRT Artificial Sequence The sequence of the BC loop of ubiquitin-binding monobody clone 411. 41 Ser Arg Leu Arg Arg 1 5 42 5 PRTArtificial Sequence The sequence of the FG loop of ubiquitin- bindingmonobody clone 411. 42 Pro Pro Trp Arg Val 1 5 43 5 PRT ArtificialSequence The sequence of the BC loop of ubiquitin- binding monobodyclone 422. 43 Ala Arg Trp Thr Leu 1 5 44 5 PRT Artificial Sequence Thesequence of the FG loop of ubiquitin- binding monobody clone 422. 44 ArgArg Trp Trp Trp 1 5 45 5 PRT Artificial Sequence The sequence of the BCloop of ubiquitin- binding monobody clone 424. 45 Gly Gln Arg Thr Phe 15 46 5 PRT Artificial Sequence The sequence of the FG loop of ubiquitin-binding monobody clone 424. 46 Arg Arg Trp Trp Ala 1 5 47 5 PRT UnknownThe sequence of the BC loop of WT from library #2. 47 Ala Val Thr ValArg 1 5 48 7 PRT Unknown The sequence of the FG loop of WT from library#2. 48 Arg Gly Asp Ser Pro Ala Ser 1 5 49 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.1. 49 Cys Asn Trp Arg Arg 1 5 507 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.1.50 Arg Ala Tyr Arg Tyr Arg Trp 1 5 51 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.2. 51 Cys Met Trp Arg Ala 1 5 527 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.2.52 Arg Trp Gly Met Leu Arg Arg 1 5 53 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.3. 53 Ala Arg Met Arg Glu 1 5 547 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.3.54 Arg Trp Leu Arg Gly Arg Tyr 1 5 55 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.4. 55 Cys Ala Arg Arg Arg 1 5 567 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.4.56 Arg Arg Ala Gly Trp Gly Trp 1 5 57 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.5. 57 Cys Asn Trp Arg Arg 1 5 587 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.5.58 Arg Ala Tyr Arg Tyr Arg Trp 1 5 59 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.6. 59 Arg Trp Arg Glu Arg 1 5 607 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.6.60 Arg His Pro Trp Thr Glu Arg 1 5 61 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.7. 61 Cys Asn Trp Arg Arg 1 5 627 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.7.62 Arg Ala Tyr Arg Tyr Arg Trp 1 5 63 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.8. 63 Glu Arg Arg Val Pro 1 5 647 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.8.64 Arg Leu Leu Leu Trp Gln Arg 1 5 65 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.9. 65 Gly Arg Gly Ala Gly 1 5 667 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.9.66 Phe Gly Ser Phe Glu Arg Arg 1 5 67 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.11. 67 Cys Arg Trp Thr Arg 1 5 687 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.11.68 Arg Arg Trp Phe Asp Gly Ala 1 5 69 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB24.12. 69 Cys Asn Trp Arg Arg 1 5 707 PRT Artificial Sequence The sequence of the FG loop of clone pLB24.12.70 Arg Ala Tyr Arg Tyr Arg Trp 1 5 71 5 PRT Unknown The sequence of theBC loop of WT from library #4. 71 Ala Val Thr Val Arg 1 5 72 5 PRTUnknown The sequence of the FG loop of WT from library #4. 72 Gly ArgGly Asp Ser 1 5 73 5 PRT Artificial Sequence The sequence of the BC loopof clone pLB25.1. 73 Gly Gln Arg Thr Phe 1 5 74 5 PRT ArtificialSequence The sequence of the FG loop of clone pLB25.1. 74 Arg Arg TrpTrp Ala 1 5 75 5 PRT Artificial Sequence The sequence of the BC loop ofclone pLB25.2. 75 Gly Gln Arg Thr Phe 1 5 76 5 PRT Artificial SequenceThe sequence of the FG loop of clone pLB25.2. 76 Arg Arg Trp Trp Ala 1 577 5 PRT Artificial Sequence The sequence of the BC loop of clonepLB25.3. 77 Gly Gln Arg Thr Phe 1 5 78 5 PRT Artificial Sequence Thesequence of the FG loop of clone pLB25.3. 78 Arg Arg Trp Trp Ala 1 5 795 PRT Artificial Sequence The sequence of the BC loop of clone pLB25.4.79 Leu Arg Tyr Arg Ser 1 5 80 5 PRT Artificial Sequence The sequence ofthe FG loop of clone pLB25.4. 80 Gly Trp Arg Trp Arg 1 5 81 5 PRTArtificial Sequence The sequence of the BC loop of clone pLB25.5. 81 GlyGln Arg Thr Phe 1 5 82 5 PRT Artificial Sequence The sequence of the FGloop of clone pLB25.5. 82 Arg Arg Trp Trp Ala 1 5 83 5 PRT ArtificialSequence The sequence of the BC loop of clone pLB25.6. 83 Gly Gln ArgThr Phe 1 5 84 5 PRT Artificial Sequence The sequence of the FG loop ofclone pLB25.6. 84 Arg Arg Trp Trp Ala 1 5 85 5 PRT Artificial SequenceThe sequence of the BC loop of clone pLB25.7. 85 Leu Arg Tyr Arg Ser 1 586 5 PRT Artificial Sequence The sequence of the FG loop of clonepLB25.7. 86 Gly Trp Arg Trp Arg 1 5 87 5 PRT Artificial Sequence Thesequence of the BC loop of clone pLB25.9. 87 Leu Arg Tyr Arg Ser 1 5 885 PRT Artificial Sequence The sequence of the FG loop of clone pLB25.9.88 Gly Trp Arg Trp Arg 1 5 89 5 PRT Artificial Sequence The sequence ofthe BC loop of clone pLB25.11. 89 Gly Gln Arg Thr Phe 1 5 90 5 PRTArtificial Sequence The sequence of the FG loop of clone pLB25.11. 90Arg Arg Trp Trp Ala 1 5 91 5 PRT Artificial Sequence The sequence of theBC loop of clone pLB25.12. 91 Leu Arg Tyr Arg Ser 1 5 92 5 PRTArtificial Sequence The sequence of the FG loop of clone pLB25.12. 92Gly Trp Arg Trp Arg 1 5 93 15 DNA Unknown The sequence of the BC loop ofWT from Table 7. 93 gcagttaccg tgcgt 15 94 5 PRT Unknown The sequence ofthe BC loop of WT from Table 7. 94 Ala Val Thr Val Arg 1 5 95 24 DNAUnknown The sequence of the FG loop of WT from Table 7. 95 ggccgtggtgacagcccagc gagc 24 96 8 PRT Unknown The sequence of the FG loop of WTfrom Table 7. 96 Gly Arg Gly Asp Ser Pro Ala Ser 1 5 97 15 DNAArtificial Sequence The sequence of the BC loop of clone 1 from Table 7.97 tcgaggttgc ggcgg 15 98 5 PRT Artificial Sequence The sequence of theBC loop of clone 1 from Table 7. 98 Ser Arg Leu Arg Arg 1 5 99 15 DNAArtificial Sequence The sequence of the FG loop of clone 1 from Table 7.99 ccgccgtgga gggtg 15 100 5 PRT Artificial Sequence The sequence of theFG loop of clone 1 from Table 7. 100 Pro Pro Trp Arg Val 1 5 101 15 DNAArtificial Sequence The sequence of the BC loop of clone 2 from Table 7.101 ggtcagcgaa ctttt 15 102 5 PRT Artificial Sequence The sequence ofthe BC loop of clone 2 from Table 7. 102 Gly Gln Arg Thr Phe 1 5 103 15DNA Artificial Sequence The sequence of the FG loop of clone 2 fromTable 7. 103 aggcggtggt gggct 15 104 5 PRT Artificial Sequence Thesequence of the FG loop of clone 2 from Table 7. 104 Arg Arg Trp Trp Ala1 5 105 15 DNA Artificial Sequence The sequence of the BC loop of clone3 from Table 7. 105 gcgaggtgga cgctt 15 106 5 PRT Artificial SequenceThe sequence of the BC loop of clone 3 from Table 7. 106 Ala Arg Trp ThrLeu 1 5 107 15 DNA Artificial Sequence The sequence of the FG loop ofclone 3 from Table 7. 107 aggcggtggt ggtgg 15 108 5 PRT ArtificialSequence The sequence of the FG loop of clone 3 from Table 7. 108 ArgArg Trp Trp Trp 1 5 109 5 PRT Artificial Sequence A solubility tail. 109Gly Lys Lys Gly Lys 1 5 110 96 PRT Artificial Sequence The synthetic Fn3gene. 110 Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala AlaThr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val ThrVal Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser ProVal Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile SerGly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val ThrGly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile AsnTyr Arg Thr 85 90 95 111 308 DNA Artificial Sequence The designed Fn3gene. 111 catatgcagg tttctgatgt tccgcgtgac ctggaagttg ttgctgcgaccccgactagc 60 ctgctgatca gctgggatgc tcctgcagtt accgtgcgtt attaccgtatcacgtacggt 120 gaaaccggtg gtaactcccc ggttcaggaa ttcactgtac ctggttccaagtctactgct 180 accatcagcg gcctgaaacc gggtgtcgac tataccatca ctgtatacgctgttactggc 240 cgtggtgaca gcccagcgag ctccaagcca atctcgatta actaccgtacctagtaactc 300 gaggatcc 308 112 96 PRT Artificial Sequence The designedFn3 gene. 112 Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val AlaAla Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala ValThr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn SerPro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr IleSer Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala ValThr Gly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser IleAsn Tyr Arg Thr 85 90 95 113 113 000 114 20 PRT Artificial Sequence Afusion protein. 114 Met Gly Ser Ser His His His His His His Ser Ser GlyLeu Val Pro 1 5 10 15 Arg Gly Ser His 20 115 10 PRT Artificial SequenceA sequence from clone Plb25.1. 115 Gly Gln Arg Thr Phe Arg Arg Trp TrpAla 1 5 10 116 10 PRT Artificial Sequence A sequence from clone Plb25.4.116 Leu Arg Tyr Arg Ser Gly Trp Arg Trp Arg 1 5 10 117 12 PRT ArtificialSequence A sequence from clone pLB24.1. 117 Cys Asn Trp Arg Arg Arg AlaTyr Arg Tyr Trp Arg 1 5 10 118 12 PRT Artificial Sequence A sequencefrom clone pLB24.3. 118 Ala Arg Met Arg Glu Arg Trp Leu Arg Gly Arg Tyr1 5 10 119 4 PRT Homo sapiens 119 Glu Ile Asp Lys 1 120 4 PRT UnknownAnti-hen egg lysozyme (HEL) antibody. 120 Arg Asp Tyr Arg 1 121 96 PRTHomo sapiens 121 Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val AlaAla Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala ValThr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn SerPro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr IleSer Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala ValThr Gly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser IleAsn Tyr Arg Thr 85 90 95

What is claimed is:
 1. A fibronectin type III (Fn3) molecule, whereinthe Fn3 comprises a stabilizing mutation as compared to a wild-type Fn3.2. The Fn3 of claim 1, wherein the stabilizing mutation comprises atleast one aspartic acid (Asp) residue that has been deleted orsubstituted with at least one other amino acid residue.
 3. The Fn3 ofclaim 2, wherein Asp 7 or Asp 23, or both, have been deleted orsubstituted with at least one other amino acid residue.
 4. The Fn3 ofclaim 3, wherein Asp 7 or Asp 23, or both, have been substituted with anasparagine (Asn) or lysine (Lys) residue.
 5. The Fn3 of claim 1, whereinthe stabilizing mutation comprises at least one glutamic acid (Glu)residue that has been deleted or substituted with at least one otheramino acid residue.
 6. The Fn3 of claim 5, wherein Glu 9 has beendeleted or substituted with at least one other amino acid residue. 7.The Fn3 of claim 6, wherein Glu 9 has been substituted with anasparagine (Asn) or lysine (Lys) residue.
 8. The Fn3 of claim 2, whereinAsp 7, Asp 23, and Glu 9 have been deleted or substituted with at leastone other amino acid residue.
 9. A fibronectin type III (Fn3)polypeptide monobody comprising a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein one or more of the monobody loop region sequences vary bydeletion, insertion or replacement of at least two amino acids from thecorresponding loop region sequences in wild-type Fn3; wherein theβ-strand domains of the monobody have at least a 50% total amino acidsequence homology to the corresponding amino acid sequence of wild-typeFn3′s β-strand domain sequences; and wherein the Fn3 comprises astabilizing mutation.
 10. An isolated nucleic acid molecule encoding theFn3 molecule of claim
 9. 11. An expression vector comprising anexpression cassette operably linked to the nucleic acid molecule ofclaim
 10. 12. A host cell comprising the vector of claim
 11. 13. Themonobody of claim 9, wherein at least one loop region is capable ofbinding to a specific binding partner (SBP) to form a polypeptide:SBPcomplex having a dissociation constant of less than 10⁻⁶ moles/liter.14. The monobody of claim 9, wherein at least one loop region is capableof catalyzing a chemical reaction with a catalyzed rate constant(k_(cat)) and an uncatalyzed rate constant (k_(uncat)) such that theratio of k_(cat)/k_(uncat) is greater than
 10. 15. The monobody of claim9, wherein one or more of the loop regions comprise amino acid residues:i) from 15 to 16 inclusive in an AB loop; ii) from 22 to 30 inclusive ina BC loop; iii) from 39 to 45 inclusive in a CD loop; iv) from 51 to 55inclusive in a DE loop; v) from 60 to 66 inclusive in an EF loop; andvi) from 76 to 87 inclusive in an FG loop.
 16. The monobody of claim 9,wherein the monobody loop region sequences vary from the wild-type Fn3loop region sequences by the deletion or replacement of at least 2 aminoacids.
 17. The monobody of claim 9, wherein the monobody loop regionsequences vary from the wild-type Fn3 loop region sequences by theinsertion of from 3 to 25 amino acids.
 18. An isolated nucleic acidmolecule encoding the polypeptide monobody of claim
 1. 19. An expressionvector comprising an expression cassette operably linked to the nucleicacid molecule of claim
 18. 20. The expression vector of claim 19,wherein the expression vector is an M13 phage-based plasmid.
 21. A hostcell comprising the vector of claim
 19. 22. A method of preparing afibronectin type III (Fn3) polypeptide monobody comprising the steps of:a) providing a DNA sequence encoding a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein at least one loop region contains a unique restriction enzymesite, and wherein at least one of the plurality of Fn3 β-strand domainsequences are more stable at neutral pH than wild-type Fn3; b) cleavingthe DNA sequence at the unique restriction site; c) inserting into therestriction site a DNA segment known to encode a peptide capable ofbinding to a specific binding partner (SBP) or a transition state analogcompound (TSAC) so as to yield a DNA molecule comprising the insertionand the DNA sequence of (a); and d) expressing the DNA molecule so as toyield polypeptide monobody.
 23. A method of preparing a fibronectin typeIII (Fn3) polypeptide monobody comprising the steps of: (a) providing areplicatable DNA sequence encoding a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein the nucleotide sequence of at least one loop region is known,and wherein at least one of the plurality of Fn3 β-strand domainsequences are more stable at neutral pH than wild-type Fn3; (b)preparing polymerase chain reaction (PCR) primers sufficientlycomplementary to the known loop sequence so as to be hybridizable underPCR conditions, wherein at least one of the primers contains a modifiednucleic acid sequence to be inserted into the DNA; (c) performingpolymerase chain reaction using the DNA sequence of (a) and the primersof (b); (d) annealing and extending the reaction products of (c) so asto yield a DNA product; and (e) expressing the polypeptide monobodyencoded by the DNA product of (d).
 24. A method of preparing afibronectin type III (Fn3) polypeptide monobody comprising the steps of:a) providing a replicatable DNA sequence encoding a plurality of Fn3β-strand domain sequences that are linked to a plurality of loop regionsequences, wherein the nucleotide sequence of at least one loop regionis known, and wherein at least one of the plurality of Fn3 β-stranddomain sequences are more stable at neutral pH than wild-type Fn3; b)performing site-directed mutagenesis of at least one loop region so asto create a DNA sequence comprising an insertion mutation; and c)expressing the polypeptide monobody encoded by the DNA sequencecomprising the insertion mutation.
 25. A kit for performing the methodof any one of claims 22-24, comprising a replicatable DNA encoding aplurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein at least one of theplurality of Fn3 β-strand domain sequences are more stable at neutral pHthan wild-type Fn3.
 26. A variegated nucleic acid library encoding Fn3polypeptide monobodies comprising a plurality of nucleic acid specieseach comprising a plurality of loop regions, wherein the species encodea plurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein one or more of the loopregion sequences vary by deletion, insertion or replacement of at leasttwo amino acids from corresponding loop region sequences in wild-typeFn3; wherein the β-strand domain sequences of the monobody have at leasta 50% total amino acid sequence homology to the corresponding amino acidsequences of β-strand domain sequences of the wild-type Fn3; and whereinthe Fn3 is more stable at neutral pH than wild-type Fn
 27. Thevariegated nucleic acid library of claim 26, wherein one or more of theloop regions encodes: i) an AB amino acid loop from residue 15 to 16inclusive; ii) a BC amino acid loop from residue 22 to 30 inclusive;iii) a CD amino acid loop from residue 39 to 45 inclusive; iv) a DEamino acid loop from residue 51 to 55 inclusive; v) an EF amino acidloop from residue 60 to 66 inclusive; and vi) an FG amino acid loop fromresidue 76 to 87 inclusive.
 28. The variegated nucleic acid library ofclaim 26, wherein the loop region sequences vary from the wild-type Fn3loop region sequences by the deletion or replacement of at least 2 aminoacids.
 29. The variegated nucleic acid library of claim 26, wherein themonobody loop region sequences vary from the wild-type Fn3 loop regionsequences by the insertion of from 3 to 25 amino acids.
 30. Thevariegated nucleic acid library of claim 26, wherein a variegatednucleic acid sequence comprising from 6 to 75 nucleic acid bases isinserted in any one of the loop regions of the species.
 31. Thevariegated nucleic acid library of claim 26, wherein the variegatedsequence is constructed so as to avoid one or more codons selected fromthe group consisting of those codons encoding cysteine or the stopcodon.
 32. The variegated nucleic acid library of claim 26, wherein thevariegated nucleic acid sequence is located in the BC loop.
 33. Thevariegated nucleic acid library of claim 26, wherein the variegatednucleic acid sequence is located in the DE loop.
 34. The variegatednucleic acid library of claim 26, wherein the variegated nucleic acidsequence is located in the FG loop.
 35. The variegated nucleic acidlibrary of claim 26, wherein the variegated nucleic acid sequence islocated in the AB loop.
 36. The variegated nucleic acid library of claim26, wherein the variegated nucleic acid sequence is located in the CDloop.
 37. The variegated nucleic acid library of claim 26, wherein thevariegated nucleic acid sequence is located in the EF loop.
 38. Apeptide display library derived from the variegated nucleic acid libraryof claim
 26. 39. A peptide display library of claim 38, wherein thepeptide is displayed on the surface of a bacteriophage or virus.
 40. Apeptide display library of claim 39, wherein the bacteriophage is M13 orfd.
 41. A method of identifying the amino acid sequence of a polypeptidemolecule capable of binding to a specific binding partner (SBP) so as toform a polypeptide:SSP complex wherein the dissociation constant of thethe polypeptide:SBP complex is less than 10⁻⁶ moles/liter, comprisingthe steps of: a) providing a peptide display library according to claim39; b) contacting the peptide display library of (a) with an immobilizedor separable SBP; c) separating the peptide:SBP complexes from the freepeptides, d) causing the replication of the separated peptides of (c) soas to result in a new peptide display library distinguished from that in(a) by having a lowered diversity and by being enriched in displayedpeptides capable of binding the SBP; e) optionally repeating steps (b),(c), and (d) with the new library of (d); and f) determining the nucleicacid sequence of the region encoding the displayed peptide of a speciesfrom (d) and deducing the peptide sequence capable of binding to theSBP.
 42. A method of preparing a variegated nucleic acid libraryencoding Fn3 polypeptide monobodies having a plurality of nucleic acidspecies each comprising a plurality of loop regions, wherein the speciesencode a plurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein one or more of the loopregion sequences vary by deletion, insertion or replacement of at leasttwo amino acids from corresponding loop region sequences in wild-typeFn3, and wherein the β-strand domain sequences of the monobody have atleast a 50% total amino acid sequence homology to the correspondingamino acid sequences of β-strand domain sequences of the wild-type Fn3,and wherein the Fn3 comprises a stabilizing mutation β-strand domain,comprising the steps of a) preparing an Fn3 polypeptide monobody havinga predetermined sequence; b) contacting the polypeptide with a specificbinding partner (SBP) so as to form a polypeptide:SSP complex whereinthe dissociation constant of the the polypeptide:SBP complex is lessthan 10⁻⁶ moles/liter; c) determining the binding structure of thepolypeptide:SBP complex by nuclear magnetic resonance spectroscopy orX-ray crystallography; and d) preparing the variegated nucleic acidlibrary, wherein the variegation is performed at positions in thenucleic acid sequence which, from the information provided in (c),result in one or more polypeptides with improved binding to the SBP. 43.A method of identifying the amino acid sequence of a polypeptidemolecule capable of catalyzing a chemical reaction with a catalyzed rateconstant, k_(cat), and an uncatalyzed rate constant, k_(uncat), suchthat the ratio of k_(cat)/k_(uncat) is greater than 10, comprising thesteps of: a) providing a peptide display library according to claim 39;b) contacting the peptide display library of (a) with an immobilized orseparable transition state analog compound (TSAC) representing theapproximate molecular transition state of the chemical reaction; c)separating the peptide:TSAC complexes from the free peptides; d) causingthe replication of the separated peptides of (c) so as to result in anew peptide display library distinguished from that in (a) by having alowered diversity and by being enriched in displayed peptides capable ofbinding the TSAC; e) optionally repeating steps (b), (c), and (d) withthe new library of (d); and f) determining the nucleic acid sequence ofthe region encoding the displayed peptide of a species from (d) andhence deducing the peptide sequence.
 44. A method of preparing avariegated nucleic acid library encoding Fn3 polypeptide monobodieshaving a plurality of nucleic acid species each comprising a pluralityof loop regions, wherein the species encode a plurality of Fn3 β-stranddomain sequences that are linked to a plurality of loop regionsequences, wherein one or more of the loop region sequences vary bydeletion, insertion or replacement of at least two amino acids fromcorresponding loop region sequences in wild-type Fn3, and wherein theβ-strand domain sequences of the monobody have at least a 50% totalamino acid sequence homology to the corresponding amino acid sequencesof β-strand domain sequences of the wild-type Fn3, and wherein the Fn3comprises a stabilizing mutation β-strand domain, comprising the stepsof a) preparing an Fn3 polypeptide monobody having a predeterminedsequence, wherein the polypeptide is capable of catalyzing a chemicalreaction with a catalyzed rate constant, k_(cat), and an uncatalyzedrate constant, k_(uncat), such that the ratio of k_(cat)/k_(uncat) isgreater than 10; b) contacting the polypeptide with an immobilized orseparable transition state analog compound (TSAC) representing theapproximate molecular transition state of the chemical reaction; c)determining the binding structure of the polypeptide:TSAC complex bynuclear magnetic resonance spectroscopy or X-ray crystallography; and d)preparing the variegated nucleic acid library, wherein the variegationis performed at positions in the nucleic acid sequence which, from theinformation provided in (c), result in one or more polypeptides withimproved binding to or stabilization of the TSAC.
 45. An isolatedpolypeptide identified by the method of claim
 41. 46. An isolatedpolypeptide identified by the method of claim
 43. 47. A kit foridentifying the amino acid sequence of a polypeptide molecule capable ofbinding to a specific binding partner (SBP) so as to form apolypeptide:SSP complex wherein the dissociation constant of the thepolypeptide: SBP complex is less than 10⁻⁶ moles/liter, comprising thepeptide display library of claim
 39. 48. A kit for identifying the aminoacid sequence of a polypeptide molecule capable of catalyzing a chemicalreaction with a catalyzed rate constant, k_(cat), and an uncatalyzedrate constant, k_(uncat), such that the ratio of k_(cat)/k_(uncat) isgreater than 10, comprising the peptide display library of claim
 39. 49.A polypeptide derived by using the kit of claim
 47. 50. A polypeptidederived by using the kit of claim 48.